Peptides for vaccine against birch allergy

ABSTRACT

The present invention relates to compositions comprising peptides for preventing or treating allergy to birch, and in particular to optimal combinations of peptides for preventing or treating said allergy.

FIELD OF THE INVENTION

The present invention relates to compositions for preventing or treatingallergy to birch.

BACKGROUND OF THE INVENTION

T-cell antigen recognition requires antigen presenting cells (APCs) topresent antigen fragments (peptides) on their cell surface inassociation with molecules of the major histocompatibility complex(MHC). T cells use their antigen specific T-cell receptors (TCRs) torecognise the antigen fragments presented by the APC. Such recognitionacts as a trigger to the immune system to generate a range of responsesto eradicate the antigen which has been recognised.

Recognition of external antigens by the immune system of an organism,such as man, can in some cases result in diseases, known as atopicconditions. Examples of the latter are the allergic diseases includingasthma, atopic dermatitis and allergic rhinitis. In this group ofdiseases, B lymphocytes generate antibodies of the IgE class (in humans)which bind externally derived antigens, which are referred to in thiscontext as allergens since these molecules elicit an allergic response.Production of allergen-specific IgE is dependent upon T lymphocyteswhich are also activated by (are specific for) the allergen.Allergen-specific IgE antibodies bind to the surface of cells such asbasophils and mast cells by virtue of the expression by these cells ofsurface receptors for IgE.

Crosslinking of surface bound IgE molecules by allergen results indegranulation of these effector cells causing release of inflammatorymediators such as histamine, 5-hydroxtryptamine and lipid mediators suchas the sulphidoleukotrienes. In addition to IgE-dependent events,certain allergic diseases such as asthma are characterised byIgE-independent events.

Allergic IgE-mediated diseases are currently treated with agents whichprovide symptomatic relief or prevention. Examples of such agents areanti-histamines, β2 agonists, and glucocorticosteroids. In addition,some IgE-mediated diseases are treated by desensitisation proceduresthat involve the periodic injection of allergen components or extracts.Desensitisation treatments may induce an IgG response that competes withIgE for allergen, or they may induce specific suppressor T cells thatblock the synthesis of IgE directed against allergen. This form oftreatment is not always effective and poses the risk of provokingserious side effects, particularly general anaphylactic shock. This canbe fatal unless recognised immediately and treated with adrenaline. Atherapeutic treatment that would decrease or eliminate the unwantedallergic-immune response to a particular allergen, without altering theimmune reactivity to other foreign antigens or triggering an allergicresponse itself would be of great benefit to allergic individuals.

Pollen allergens are recognised as a major cause of allergic diseases inhumans and animals, including asthma, allergic rhinitis and allergicdermatitis. At least 10% of the population of the USA suffers frompollen allergies at various times and to varying extents. Proteinspresent in tree pollen, in particular from trees of the order Fagales,for example birch, alder, hazel, hornbeam and oak, are particularlyimportant. Of these species, birch pollen allergens are the mostfrequent initiators of allergic responses to tree pollen (Jarolim et al:Allergy 1989; 44(6):385-95). For example, approximately 25% of hayfeversufferers are responsive to birch pollen. Hayfever is the common termfor a form of seasonal allergy characterised by sneezing, runny nose anditching eyes. Allergy to tree pollen is most problematic during thespring months, with the birch pollen season typically occurring aroundApril (in the northern hemisphere). However, some related types of treesuch as alder and hazel can release airborne pollen as early as January(northern hemisphere). These are followed by elm, willow and ash inMarch, with oak in late April and early May.

It has been calculated that for adults in the United States, hayfever isthe 5th leading chronic disease and a major cause of work absenteeism,resulting in nearly 4 million missed or lost workdays each year,resulting in a total cost of more than $700 million in total lostproductivity. Allergies are also the most frequently reported chroniccondition in children, limiting activities for more than 40% of them.Each year, allergies account for more than 17 million outpatient officevisits in the United States; seasonal allergies such as hayfever accountfor more than half of these allergy visits.

A therapeutic or preventative treatment would therefore be of greatbenefit to humans that suffer or are at risk of suffering from treeallergy.

SUMMARY OF THE INVENTION

The present inventors have discovered that certain peptide fragmentsderived from the major allergens in the pollens of birch species areuseful in desensitising individuals to these allergens. Peptidefragments derived from Bet v2, Bet v1, Bet v3, Bet v4, Bet v6 and Bet v7of birch (family name: Betulucaea) are particularly useful.

The peptides of the invention were selected as MHC class II-binding Tcell epitopes through use of in silico analysis to predict peptide-MHCinteractions and MHC class II binding assays. Additional epitopes wereidentified by homology.

A difficulty associated with approaches to desensitisation based onpeptide immunisation lies in how to select an appropriate size andregion of the allergen as the basis for the peptide to be used forimmunisation. The size of the peptide of choice is crucial. If thepeptide is too small, the vaccine would not be effective in inducing animmunological response. If the peptides are too large, or if the wholeantigen is introduced into an individual, there is the risk of inducingadverse reactions, such as anaphylaxis, which may be fatal.

The polypeptides of the invention have been selected to retain T cellspecificity whilst being small enough in size to not possess significanttertiary structure that would enable them to retain the conformation ofan IgE-binding epitope of the whole molecule. The polypeptides of theinvention therefore do not induce significant crosslinking of adjacentspecific IgE molecules on cells such as mast cells and basophils andconsequently do not cause significant histamine release.

An advantage of the invention is the ability of the peptides to broadlytarget Major Histocompatibility Complex (MHC) molecules. T cellreceptors (TCRs) are highly variable in their specificity. Variabilityis generated, as with antibody molecules, through gene recombinationevents within the cell. TCRs recognise antigen in the form of shortpeptides bound to molecules encoded by the genes of the MajorHistocompatibility Complex (MHC). These gene products are the samemolecules that give rise to “tissue types” used in transplantation andare also referred to as Human Leukocyte Antigen molecules (HLAs) whichterms may be used interchangeably. Individual MHC molecules possesspeptide binding grooves which, due to their shape and charge are onlycapable of binding a limited group of peptides. The peptides bound byone MHC molecule may not necessarily be bound by other MHC molecules.

When a protein molecule such as an antigen or allergen is taken up byantigen presenting cells such as B lymphocytes, dendritic cells,monocytes and macrophages, the molecule is enzymatically degraded withinthe cell. The process of degradation gives rise to peptide fragments ofthe molecule which, if they are of the appropriate size, charge andshape, may then bind within the peptide binding groove of certain MHCmolecules and be subsequently displayed upon the surface of antigenpresenting cells. If the peptide/MHC complexes are present upon theantigen presenting cell surface in sufficient numbers they may thenactivate T cells which bear the appropriate peptide/MHC-specific T cellreceptors.

Due to the polymorphic nature of the MHC, individuals in an outbredpopulation such as man will express different combinations of MHCmolecules on their cell surfaces. Since different MHC molecules can binddifferent peptides from the same molecule based on the size, charge andshape of the peptide, different individuals will display a differentrepertoire of peptides bound to their MHC molecules. Identification ofuniversal MHC-binding peptide epitopes in an outbred population such asman is more difficult than in inbred animals (such as certain strains oflaboratory mice). On the basis of differential MHC expression betweenindividuals and the inherent differences in peptide binding andpresentation which this brings, it is unlikely that a single peptide canbe identified which will be of use for desensitisation therapy in man.

The peptides of the invention, however, provide a broad coverage ofefficacy over the human population by targeting multiple different MHCmolecules. A vaccine formulated with a peptide of the invention wouldtherefore have broad utility. Accordingly, the present inventionprovides a composition suitable for use in preventing or treatingallergy to birch pollen by tolerisation comprising:

i) at least one of the polypeptides of SEQ ID NO: 74 (BIR12B;AKYMVIQGEPGRVIRGK), SEQ ID NO: 72 (BIR11; FPQFKPQEITGIMK), SEQ ID NO: 71(BIR10; GSVWAQSSSFPQFK), SEQ ID NO: 73 (BIR12A; PTGMFVAGAKYMVIQGR), SEQID NO: 75 (BIR13; IKYMVIQGEAGAVIRGK and SEQ ID NO: 76 (BIR14;EAGAVIRGKKGSGGIT), or a variant of any thereof, andii) at least one of the polypeptides of SEQ ID NO: 53 (Bir02J;PAARMFKAFILEGDKLVPK), SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK), SEQ IDNO: 54 (Bir04; PGTIKKISFPEGFPFKYV), SEQ ID NO: 67 (Bir09;ETLLRAVESYLLAHSDAY), SEQ ID NO: 60 (BIR07; SNEEKIVATPDGGSILK), and SEQID NO: 63 (Bir07C; SNEIKIVATPEGGSILK), or a variant of any thereof,wherein said variant is:

-   -   I) a longer polypeptide of up to 30 amino acids in length which        comprises the sequence of the corresponding polypeptide        specified in (i) or (ii), or    -   II) a polypeptide of 9 to 30 amino acids in length which        comprises a sequence that has at least 65% homology to the        sequence of the corresponding polypeptide specified in (i) or        (ii), which sequence is capable of tolerising to said        corresponding polypeptide; or    -   III) a polypeptide of length 9 to 30 amino acids which comprises        a sequence of, or a sequence that has at least 65% homology to,        at least 9 contiguous amino acids of the sequence of the        corresponding polypeptide specified in (i) or (ii), which        sequence of at least 9 contiguous amino acids or homologous        sequence is capable of tolerising to said corresponding        polypeptide.        Also provided is a composition suitable for use in preventing or        treating allergy to birch pollen by tolerisation comprising at        least three different polypeptides, selected from:        (a) Bir12B (AKYMVIQGEPGRVIRGK), or a variant thereof;        (b) Bir02J (PAARMFKAFILEGDKLVPK), or a variant thereof;        (c) Bir01I (FNYETETTSVIPAARK) or a variant thereof;        (d) Bir04 (PGTIKKISFPEGFPFKYV) or a variant thereof;        (e) Bir09 (ETLLRAVESYLLAHSDAY) or a variant thereof;        (f) Bir16A (AERERIFKRFDANGEGK) or a variant thereof;        (g) Bir07 (SNEIKIVATPDGGSILK) or a variant thereof;        (h) Bir07C (SNEIKIVATPEGGSILK) or a variant thereof;        (i) Bir011 (FPQFKPQEITGIMK) or a variant thereof;        (j) Bir15 (SLNTLRLRRIFDLFDK) or a variant thereof;        wherein said variant is:    -   I) a longer polypeptide of up to 30 amino acids in length which        comprises the sequence of the corresponding polypeptide        specified in (a) to (j), or    -   II) a polypeptide of 9 to 30 amino acids in length which        comprises a sequence that has at least 65% homology to the        sequence of the corresponding polypeptide specified in (a) to        (j), which sequence is capable of tolerising to said        corresponding polypeptide; or    -   III) a polypeptide of length 9 to 30 amino acids which comprises        a sequence of, or a sequence that has at least 65% homology to,        at least 9 contiguous amino acids of the sequence of the        corresponding polypeptide specified in (a) to (j), which        sequence of at least 9 contiguous amino acids or homologous        sequence is capable of tolerising to said corresponding        polypeptide.

Description of the Sequences Mentioned Herein

SEQ ID NOS: 1 to 80 provide the polypeptide sequences of the inventionas set out in Tables 1 to 8. SEQ ID NOS: 1 to 34 and 45 to 70 correspondto peptides derived from Bet v1. SEQ ID NOS: 71 to 76 correspond topeptides derived from Bet v2. SEQ ID NOS: 35, 36 and 77 correspond topeptides derived from Bet v3. SEQ ID NOS: 37 to 39, 78 and 79 correspondto peptides derived from Bet v4. SEQ ID NOS: 40 to 43 and 80 correspondto peptides derived from Bet v6. SEQ ID NO: 44 corresponds to a peptidederived from Bet v7.

DETAILED DESCRIPTION OF THE INVENTION

The invention concerns peptides which can be used in tolerisation. Suchpeptides may comprise, consist of, or consist essentially of thesequences shown in any of SEQ ID NOS: 1 to 80. Variants of thesespecific peptides may also be used. The variants may comprise, consistof, or consist essentially of sequences which are fragments of eitherany of SEQ ID NOS: 1 to 80 or homologues of any of SEQ ID NOS: 1 to 80.

The invention also provides products and formulations comprising thepolypeptides of the invention and compositions, products and vectorscomprising polynucleotides capable of expressing the polypeptides of theinvention for use in preventing or treating birch allergy bytolerisation. Such tolerisation will typically be to an epitope (forexample a MHC class II-binding T cell epitope) present in any of SEQ IDNOS: 1 to 80.

Tree Species

Species of tree from the family Betulaceae, commonly known as birch, areresponsible for a high proportion of tree allergy worldwide,particularly allergies associated with tree pollen, such as hayfever.Other important tree species include alder, hazel, hornbeam and oak.

Birch trees, for example Silver Birch (Betula pendula), tolerate a widerange of habitats, with soil pH from approximately 3.5 to approximately7. They are native to most of Europe and parts of Asia, but are commonthroughout the world, being found in the temperate, boreal, and arcticzones of the Northern Hemisphere, especially in Canada and other partsof North America. Birch trees typically flower between April and May(Northern Hemisphere).

Peptide Fragments of Birch Pollen Allergens

The present inventors have identified the regions in certain birchpollen allergen proteins which comprise MHC Class II-binding T cellepitopes. The present inventors have also shown that regionscorresponding to MHC Class II-binding T cell epitopes within the majorbirch pollen allergens are highly conserved between different isoformsof said allergens. Based on this information, peptides derived from therelevant regions of each protein are suitable for preventing or treatingbirch allergy by tolerisation to all isoforms of that protein.

The peptides of the invention are derived directly or by homology fromthe protein allergens Bet v2 (SEQ ID NOS: 71 to 76), Bet v1 (SEQ ID NOS:1 to 34 and 45 to 70), Bet v3 (SEQ ID NOS: 35, 36 and 77), Bet v4 (SEQID NOS: 37 to 39, 78 and 79), Bet v6 (SEQ ID NOS: 40 to 43 and 80) andBet v7 (SEQ ID NO: 44). The terms “peptide” and “polypeptide” are usedinterchangeably herein. The above proteins are also referred to hereinas “the allergens”. Tables 1 to 7 set out the sequences of the peptidesof the invention (SEQ ID NOS: 1 to 80), indicating the parent proteinfrom which each peptide derives. The composition of the inventioncomprises at least one polypeptide selected from SEQ ID NOS: 1 to 80 ora variant of any thereof.

In other words, the invention provides a composition for use in theprevention or treatment of birch allergy by tolerisation comprising atleast three, preferably at least four different polypeptides selectedfrom any of SEQ ID NOS: 1 to 80, or a variant of any thereof. It ispreferred that none of the selected polypeptides are variants of thesame original sequence defined by any one of SEQ ID NOS: 1 to 80. Inother words, it is preferred that each of the three or four polypeptidesare different original baseline sequences defined by any one of SEQ IDNOS: 1 to 80, or are variants of different original baseline sequencesdefined by any one of SEQ ID NOS: 1 to 80.

Preferably, the composition will comprise polypeptides which derive frommore than one allergen. For example, the composition may comprise one ormore polypeptides or variants thereof derived from Bet v 2 and one ormore polypeptides or variants thereof derived from Bet v 1. Additionalpolypeptides may optionally be included which derive from Bet v 3, Bet v4, Bet v 6 and/or Bet v 7. Accordingly, in some embodiments, thecomposition comprises

i) at least one of the polypeptides of SEQ ID NO: 74, 72, 71, 73, 75 and76 (which are derived from Bet v 2), or a variant of any thereof asdefined herein; andii) at least one of the polypeptides of SEQ ID NOS: 1 to 34 and 45 to 70(which are derived from Bet v 1), or a variant of any thereof; andoptionallyiii) at least one of the polypeptides of:(a) SEQ ID NOs: 35, 36 and 77 (which are derived from Bet v 3), or avariant of any thereof as defined herein; and/or(b) SEQ ID NOs: 37 to 39, 78 and 79 (which derived from Bet v 4), or avariant of any thereof as defined herein; and/or(c) SEQ ID NOs: 40 to 43 and 80 (which are derived from Bet v 6), or avariant of any thereof as defined herein; and/or(d) SEQ ID NO: 44 (which is derived from Bet v 7), or a variant thereofas defined herein.

The composition may thus comprise any combination of one or morepolypeptides selected from group (i), one or more polypeptides selectedfrom group (ii) and optionally one or more polypeptides from group (iii)(a) to (d) as defined above. Groups (i), (ii) and (iii) (a) to (d)correspond to peptides derived from different Bet allergens, asdescribed above. Combining polypeptides derived from different Betallergens may allow for broad coverage of birch pollen allergy observedin the general population by providing tolerising epitopes from morethan one birch pollen allergen.

Non-limiting examples of compositions selected as defined above include:

One, two or more polypeptides selected from SEQ ID NO: 74, 72, 71, 73,75 and 76 or variants of any thereof, at least one polypeptide selectedfrom group (ii) or variant of any thereof, and optionally at least onepolypeptide or variant thereof selected from groups (iii) (a) and/or(b); orOne, two or more polypeptides selected from SEQ ID NO: 74, 72, 71, 73,75 and 76 or variants of any thereof and two, three, four or fivepolypeptides selected from group (ii) or variants of any thereof, andoptionally at least one polypeptide or variant thereof selected fromgroups (iii) (a) and/or (b); orOne, two or more polypeptides selected from SEQ ID NO: 74, 72, 71, 73,75 and 76 or variants of any thereof and two, three, four or fivepolypeptides selected from group (ii) or variants of any thereof, and atleast one polypeptide from group (iii) b).

In one embodiment, the composition comprises:

(i) at least one of the polypeptides of SEQ ID NO: 74 (BIR12B;AKYMVIQGEPGRVIRGK), SEQ ID NO: 72 (BIR11; FPQFKPQEITGIMK), SEQ ID NO: 71(BIR10; GSVWAQSSSFPQFK), SEQ ID NO: 73 (BIR12A; PTGMFVAGAKYMVIQGR), SEQID NO: 75 (BIR13; IKYMVIQGEAGAVIRGK and SEQ ID NO: 76 (BIR14;EAGAVIRGKKGSGGIT), or a variant of any thereof, andii) at least one of the polypeptides of SEQ ID NO: 53 (Bir02J;PAARMFKAFILEGDKLVPK), SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK), SEQ IDNO: 54 (Bir04; PGTIKKISFPEGFPFKYV), SEQ ID NO: 67 (Bir09;ETLLRAVESYLLAHSDAY), SEQ ID NO: 60 (BIR07; SNEIKIVATPDGGSILK), and SEQID NO: 63 (Bir07C; SNEIKIVATPEGGSILK), or a variant of any thereof. Inanother embodiment, the composition further comprises at least oneadditional polypeptide of (i) or (ii) or variant thereof not selectedabove. In another embodiment, the composition further comprises at leastone additional polypeptide of SEQ ID NO: 77 (BIR15; SLNTLRLRRIFDLFDK) orSEQ ID NO: 78 (BIR16A; AERERIFKRFDANGEGK), or a variant of any thereof.In a preferred embodiment, the composition comprises:(a) the polypeptide Bir12B (AKYMVIQGEPGRVIRGK), or a variant thereof;(b) the polypeptide Bir02J (PAARMFKAFILEGDKLVPK), or a variant thereof;and(c) the polypeptide Bir01I (FNYETETTSVIPAARK) or a variant thereof;

In a particularly preferred embodiment, the composition comprises thepolypeptide Bir12B (AKYMVIQGEPGRVIRGK) or a variant thereof, thepolypeptide Bir02J (PAARMFKAFILEGDKLVPK) or a variant thereof, thepolypeptide Bir01I (FNYETETTSVIPAARK) or a variant thereof, thepolypeptide Bir04 (PGTIKKISFPEGFPFKYV) or a variant thereof, thepolypeptide Bir09 (ETLLRAVESYLLAHSDAY) or a variant thereof, thepolypeptide Bir07C (SNEIKIVATPEGGSILK) or a variant thereof, and thepolypeptide Bir16A (AERERIFKRFDANGEGK) or a variant thereof, andoptionally no further polypeptides.

In a further particularly preferred embodiment, the compositioncomprises the polypeptide Bir12B (AKYMVIQGEPGRVIRGK) or a variantthereof, the polypeptide Bir02J (PAARMFKAFILEGDKLVPK) or a variantthereof, the polypeptide Bir01I (FNYETETTSVIPAARK) or a variant thereof,the polypeptide Bir04 (PGTIKKISFPEGFPFKYV) or a variant thereof, thepolypeptide Bir07C (SNEIKIVATPEGGSILK) or a variant thereof, thepolypeptide Bir16A (AERERIFKRFDANGEGK) or a variant thereof, and thepolypeptide Bir09B (KEMGETLLRAVESYLLAHS) or a variant thereof, andoptionally no further polypeptides.

In a further particularly preferred embodiment, the compositioncomprises the polypeptide Bir12B (AKYMVIQGEPGRVIRGK) or a variantthereof, the polypeptide Bir02J (PAARMFKAFMEGDKLVPK) or a variantthereof, the polypeptide Bir01I (FNYETETTSVIPAARK) or a variant thereof,the polypeptide Bir04 (PGTIKKISFPEGFPFKYV) or a variant thereof, thepolypeptide Bir07C (SNEIKIVATPEGGSILK) or a variant thereof, and thepolypeptide Bir16A (AERERIFKRFDANGEGK) or a variant thereof, andoptionally no further polypeptides.

The invention also provides a product comprising a peptide, variant orcomposition according to the invention. The invention provides a productcomprising:

i) at least one of the polypeptides of SEQ ID NO: 74 (BIR12B;AKYMVIQGEPGRVIRGK), SEQ ID NO: 72 (BIR11; FPQFKPQEITGIMK), SEQ ID NO: 71(BIR10; GSVWAQSSSFPQFK), SEQ ID NO: 73 (BIR12A; PTGMFVAGAKYMVIQGR), SEQID NO: 75 (BIR13; IKYMVIQGEAGAVIRGK and SEQ ID NO: 76 (BIR14;EAGAVIRGKKGSGGIT), or a variant of any thereof as defined in (I) to(III), andii) at least one of the polypeptides of SEQ ID NO: 53 (Bir02J;PAARMFKAFILEGDKLVPK), SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK), SEQ IDNO: 54 (Bir04; PGTIKKISFPEGFPFKYV), SEQ ID NO: 67 (Bir09;ETLLRAVESYLLAHSDAY), SEQ ID NO: 60 (BIR07; SNEIKIVATPDGGSILK), and SEQID NO: 63 (Bir07C; SNEIKIVATPEGGSILK), or a variant of any thereof asdefined in (I) to (III), wherein each different polypeptide is forsimultaneous, separate or sequential use in preventing or treatingallergy to birch pollen by tolerisation.

Variants of the polypeptides of SEQ ID NOS: 1 to 80 are mentionedherein. A variant of any of SEQ ID NOS: 1 to 80 will typically befunctional. By functional it is meant that the variant is one which:

(a) comprises or consists of a sequence which binds to the same MHCclass II molecule as the corresponding polypeptide of SEQ ID NOS: 1 to80; and/or(b) comprises or consists of a sequence which is recognised by a T cellwhich recognises the corresponding polypeptide of SEQ ID NOS: 1 to 80;and/or(c) is capable of inducing a late phase response in an individual withbirch allergy; and/or(d) is capable of tolerising an individual to the correspondingpolypeptide.

Recognition by a T cell may be tested by measuring the ability of apeptide or variant to induce T cell proliferation in a sample of Tcells. The induction of a late phase response may also be tested in thisway when the sample of T cells is taken from an individual with birchallergy. Methods of testing the induction of T cell proliferation arewell known in the art and one such method is exemplified in Example 8.

Variants of SEQ ID NOS: 1 to 80 may be fragments derived by truncation,e.g. by removal of one or more amino acids from the N and/or C-terminalends of a polypeptide. Fragments may also be generated by one or moreinternal deletions, provided that the core 9 amino acids that makes upthe T cell epitope is not substantially disrupted.

For example, a variant of SEQ ID NO: 1 may comprise a fragment of SEQ IDNO: 1, i.e. a shorter sequence. This may include a deletion of one, two,three or four amino acids from the N-terminal end of SEQ ID NO: 1 orfrom the C-terminal end of SEQ ID NO: 1. Such deletions may be made fromboth ends of SEQ ID NO: 1.

A variant of SEQ ID NO: 1 may include additional amino acids (forexample from the sequence of the parent protein from which the peptidederives) extending beyond the end(s) of SEQ ID NO: 1. A variant of apolypeptide may typically be a longer polypeptide of up to 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length which comprisesthe sequence of the corresponding polypeptide of SEQ ID NOS: 1 to 80.

A variant may include a combination of the deletions and additionsdiscussed above. For example, amino acids may be deleted from one end ofSEQ ID NO: 1, but additional amino acids from the full length parentprotein sequence may be added at the other end of SEQ ID NO: 1. The samediscussion of variants above also applies to SEQ ID NOS: 2 to 80.

A variant may alternatively be a polypeptide of 9 to 30, 11 to 20 or 13to 17 amino acids in length which comprises a sequence that has at least65% sequence identity to the sequence of the corresponding polypeptideof SEQ ID NOS: 1 to 80. More preferably a suitable variant may compriseat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% amino acid identity to the correspondingpolypeptide of SEQ ID NOS: 1 to 80.

A variant may be a polypeptide of length 9 to 30, 11 to 20 or 13 to 17amino acids which comprises a sequence of, or a sequence that has, atleast 65% sequence identity to at least 9 (for example at least 10, 11,12 or 13) or more contiguous amino acids of the sequence of thecorresponding polypeptide of SEQ ID NOS: 1 to 80. These contiguous aminoacids may typically comprise a MHC class II epitope, for example whichbinds to any of the MHC molecules mentioned herein.

A variant peptide may include one or more amino acid substitutions fromthe amino acid sequence of any of SEQ ID NOS: 1 to 80 or a fragmentthereof. A variant peptide may comprise sequence having at least 65%sequence identity to at least 9 or more contiguous amino acids in any ofSEQ ID NOS: 1 to 80. More preferably a suitable variant may comprise atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% amino acid identity to at least 9 contiguousamino acids of any of SEQ ID NOS: 1 to 80. This level of amino acididentity may be seen at any section of the peptide, although it ispreferably the core region. The level of amino acid identity is over atleast 9 contiguous amino acids but it may be at least 10, 11, 12, 13,14, 15 or at least 16 or 17 amino acids, depending on the size of thepeptides of comparison. Accordingly, any of the above-specified levelsof identity may be across the entire length of sequence.

In connection with amino acid sequences, “sequence identity” refers tosequences which have the stated value when assessed using ClustalW(Thompson et al., 1994, supra) with the following parameters:

Pairwise alignment parameters—Method: accurate, Matrix: PAM, Gap openpenalty: 10.00, Gap extension penalty: 0.10; Multiple alignmentparameters—Matrix: PAM, Gap open penalty: 10.00, % identity for delay:30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix:no, Gap extension penalty: 0.20, Residue-specific gap penalties: on,Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR. Sequenceidentity at a particular residue is intended to include identicalresidues which have simply been derivatized.

A variant peptide may comprise 1, 2, 3, 4, 5 or more, or up to 10 aminoacid substitutions from any of SEQ ID NOS: 1 to 80. Substitutionvariants preferably involve the replacement of one or more amino acidswith the same number of amino acids and making conservative amino acidsubstitutions. For example, an amino acid may be substituted with analternative amino acid having similar properties, for example, anotherbasic amino acid, another acidic amino acid, another neutral amino acid,another charged amino acid, another hydrophilic amino acid, anotherhydrophobic amino acid, another polar amino acid, another aromatic aminoacid or another aliphatic amino acid. Some properties of the 20 mainamino acids which can be used to select suitable substituents are asfollows:

Ala aliphatic, hydrophobic, Met hydrophobic, neutral neutral Cys polar,hydrophobic, Asn polar, hydrophilic, neutral neutral Asp polar,hydrophilic, Pro hydrophobic, neutral charged (−) Glu polar,hydrophilic, Gln polar, hydrophilic, charged (−) neutral Phe aromatic,hydrophobic, Arg polar, hydrophilic, neutral charged (+) Gly aliphatic,neutral Ser polar, hydrophilic, neutral His aromatic, polar,hydrophilic, Thr polar, hydrophilic, charged (+) neutral Ile aliphatic,hydrophobic, Val aliphatic, hydrophobic, neutral neutral Lys polar,hydrophilic, Trp aromatic, hydrophobic, charged(+) neutral Leualiphatic, hydrophobic, Tyr aromatic, polar, neutral hydrophobic

Further variants include those in which instead of the naturallyoccurring amino acid the amino acid which appears in the sequence is astructural analog thereof. Amino acids used in the sequences may also bemodified, e.g. labelled, providing the function of the peptide is notsignificantly adversely affected.

Where the peptide has a sequence that varies from the sequence of any ofSEQ ID NOS: 1 to 80 or a fragment thereof, the substitutions may occuracross the full length of the sequence, within the sequence of any ofSEQ ID NOS: 1 to 80 or outside the sequence of any of SEQ ID NOS: 1 to80. For example, the variations described herein, such as additions,deletions, substitutions and modifications, may occur within thesequence of any of SEQ ID NOS: 1 to 80. A variant peptide may compriseor consist essentially of the amino acid sequence of any of SEQ ID NOS:1 to 80 in which one, two, three, four or more amino acid substitutionshave been made. A variant peptide may comprise a fragment of the parentprotein that is larger than any of SEQ ID NOS: 1 to 80. In thisembodiment, the variations described herein, such as substitutions andmodifications, may occur within and/or outside the sequence of any ofSEQ ID NOS: 1 to 80. For example, one or more positively chargedresidues may be added at the N and/or C terminus of the native sequenceof the peptide of any of SEQ ID NOS: 1 to 80.

The variant peptides of the invention are 9 to 30 amino acids in lengthinclusive. Preferably, they may be from 9 to 20 or more preferably 13 to17 amino acids in length. The peptides may be the same length as thepeptide sequences in any one of SEQ ID NOS: 1 to 80.

The peptides may be chemically derived from the polypeptide allergen,for example by proteolytic cleavage or can be derived in an intellectualsense from the polypeptide allergen, for example by making use of theamino acid sequence of the polypeptide allergen and synthesisingpeptides based on the sequence. Peptides may be synthesised usingmethods well known in the art.

The term “peptide” includes not only molecules in which amino acidresidues are joined by peptide (—CO—NH—) linkages but also molecules inwhich the peptide bond is reversed. Such retro-inverso peptidomimeticsmay be made using methods known in the art, for example such as thosedescribed in Meziere et al (1997) 3. Immunol. 159, 3230-3237. Thisapproach involves making pseudopeptides containing changes involving thebackbone, and not the orientation of side chains. Meziere et al (1997)show that, at least for MHC class II and T helper cell responses, thesepseudopeptides are useful. Retro-inverse peptides, which contain NH—CObonds instead of CO—NH peptide bonds, are much more resistant toproteolysis.

Similarly, the peptide bond may be dispensed with altogether providedthat an appropriate linker moiety which retains the spacing between thecarbon atoms of the amino acid residues is used; it is particularlypreferred if the linker moiety has substantially the same chargedistribution and substantially the same planarity as a peptide bond. Itwill also be appreciated that the peptide may conveniently be blocked atits N- or C-terminus so as to help reduce susceptibility toexoproteolytic digestion. For example, the N-terminal amino group of thepeptides may be protected by reacting with a carboxylic acid and theC-terminal carboxyl group of the peptide may be protected by reactingwith an amine. Other examples of modifications include glycosylation andphosphorylation. Another potential modification is that hydrogens on theside chain amines of R or K may be replaced with methylene groups(—NH₂→—NH(Me) or —N(Me)₂).

Analogues of peptides according to the invention may also includepeptide variants that increase or decrease the peptide's half-life invivo. Examples of analogues capable of increasing the half-life ofpeptides used according to the invention include peptoid analogues ofthe peptides, D-amino acid derivatives of the peptides, andpeptide-peptoid hybrids. A further embodiment of the variantpolypeptides used according to the invention comprises D-amino acidforms of the polypeptide. The preparation of polypeptides using D-aminoacids rather than L-amino acids greatly decreases any unwanted breakdownof such an agent by normal metabolic processes, decreasing the amountsof agent which needs to be administered, along with the frequency of itsadministration.

The peptides provided by the present invention may be derived fromsplice variants of the parent proteins encoded by mRNA generated byalternative splicing of the primary transcripts encoding the parentprotein chains. The peptides may also be derived from amino acidmutants, glycosylation variants and other covalent derivatives of theparent proteins which retain at least an MHC-binding property of theallergens. Exemplary derivatives include molecules wherein the peptidesof the invention are covalently modified by substitution, chemical,enzymatic, or other appropriate means with a moiety other than anaturally occurring amino acid. Further included are naturally occurringvariants of the parent proteins found in different mites. Such a variantmay be encoded by an allelic variant or represent an alternativesplicing variant.

Variants as described above may be prepared during synthesis of thepeptide or by post-production modification, or when the peptide is inrecombinant form using the known techniques of site-directedmutagenesis, random mutagenesis, or enzymatic cleavage and/or ligationof nucleic acids.

In any of the embodiments of the invention, typical examples of variantsas described herein may be as follows:

a variant of Bir01I is Bir01F (FNYETEATSVIPAARK), Bir01G(FNYEIEATSVIPAARK) or Bir01H (FNYEIETTSVIPAARK); and/or

a variant of Bir02J is Bir02E (PAARLFKAFILEGDTLIPK), Bir02G(PAARLFKAFILEGDNLIPK), Bir02I (PAARMFKAFILD) or Bir02D(PAARMFKAFILDGDKLVPK); and/or

a variant of Bir09 is selected from Bir09A (GETLLRAVESYLLAHS), Bir09B(KEMGETLLRAVESYLLAHS) or Bir09C (KEKGETLLRAVESYLLAHS); and/or

a variant of Bir16B is Bir16A (AERERIFKRFDANGEGK).

It will be understood that SEQ ID NOS: 1 to 80 are polypeptide sequenceswhich comprise a T cell epitope that consists of a core of typically 9amino acids, which are the minimal essential sequence required for MHCclass II binding. However, the polypeptides of SEQ ID NOS: 1 to 80 mayalso comprise additional residues flanking the core. The peptides maytherefore comprise a region containing a T cell epitope, in which someresidues may be modified without affecting the function of the epitope.Thus, for example, the sequences of any of SEQ ID NOS: 1 to 80 may bealtered to improve their solubility, and accordingly a variant of any ofSEQ ID NOS: 1 to 80 will preferably be more soluble than thecorresponding polypeptide of SEQ ID NOS: 1 to 80 under equivalentconditions. Methods for evaluating the solubility of peptides are wellknown in the art and one such method is exemplified in Example 9.

Improved solubility is advantageous for the tolerisation of subjects toallergens from which the peptides of the invention derive, sinceadministration of poorly soluble agents to subjects causes undesirable,non-tolerising inflammatory responses. The solubility of the peptidesmay be improved by altering the residues which flank the regioncontaining a T cell epitope. A peptide of the invention may beengineered to be more soluble such that it comprises:

-   i) N terminal to the residues of the peptide which flank a T cell    epitope: one to six contiguous amino acids corresponding to the one    to six contiguous amino acids immediately N terminal to said    residues in the sequence of the protein from which the peptide    derives; and/or-   ii) C terminal to the residues of the peptide which flank a T cell    epitope: one to six contiguous amino acids corresponding to the one    to six contiguous amino acids immediately C terminal to the said    residues in the sequence of the protein from which the peptide    derives; or-   iii) both N and C terminal to the residues of the peptide which    flank a T cell epitope, at least one amino acid selected from    arginine, lysine, histidine, glutamate and aspartate.

Optionally, the peptides may additionally be engineered to be moresoluble such that:

i) any cysteine residues in the native sequence of the peptide arereplaced with serine or 2-aminobutyric acid; and/orii) any hydrophobic residues in the up to three amino acids at the N orC terminus of the native sequence of the peptide, which are notcomprised in a T cell epitope, are deleted; and/oriii) any two consecutive amino acids comprising the sequence Asp-Gly inthe up to four amino acids at the N or C terminus of the native sequenceof the peptide, which are not comprised in a T cell epitope, aredeleted; and/oriv) one or more positively charged residues are added at the N and/or Cterminus of the native sequence of the peptide.

Preferably the peptides and variants of the invention are capable ofcausing T cell proliferation in at least 20% of samples of T cells,wherein each sample is obtained from different birch allergicindividuals in the population. The compositions of the invention arepreferably capable of inducing T cell proliferation in 30% or moresamples of T cells obtained from of a panel of birch allergicindividuals. More preferably, the compositions are capable of inducing Tcell proliferation in 35% or more, 40% or more, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, or 90% or more of samples obtained from sensitizedindividuals in a panel. The number of individuals in a panel of birchallergic individuals may be any number greater than one, for example atleast 2, 3, 5, 10, 15, 20, 30, 50, 80, or at least 100 individuals.

It is preferred if the peptides, variants and compositions of theinvention cause T cell proliferation, but do not lead to the release ofhistamine from enriched basophils or mast cell preparations from asensitised individual. There may be some histamine release, butpreferably the peptides, variants and compositions do not causesignificant amounts of histamine to be released. Significant histaminerelease may be considered to be the release of 20% or more of the totalavailable leukocyte histamine when a sample of leukocytes from anindividual is stimulated with a composition in vitro. A peptide, variantor composition of the invention preferably causes the release of lessthan 5%, less than 4%, less than 3%, less than 2% or less than 1% of thetotal available leukocyte histamine when a sample of leukocytes from anindividual is stimulated with a composition in vitro. A normalindividual typically has an approximate leukocyte histamine content of150 ng/10⁷ cells.

Suitable peptides or variants capable of binding to TCRs may be derivedempirically or selected according to known criteria. Within a singlepeptide there are certain residues which contribute to binding withinthe MHC antigen binding groove and other residues which interact withhypervariable regions of the T cell receptor (Allen et al (1987) Nature327: 713-5).

Within the residues contributing to T cell receptor interaction, ahierarchy has been demonstrated which pertains to dependency of T cellactivation upon substitution of a given peptide residue. Using peptideswhich have had one or more T cell receptor contact residues substitutedwith a different amino acid, several groups have demonstrated profoundeffects upon the process of T cell activation. Evavold & Allen (1991)Nature 252: 1308-10) demonstrated the dissociation of T cellproliferation and cytokine production. In this in vitro model, a T cellclone specific for residues 64-76 of haemoglobin (in the context ofI-E^(k)), was challenged with a peptide analogue in which a conservativesubstitution of aspartic acid for glutamic acid had been made. Thissubstitution did not significantly interfere with the capacity of theanalogue to bind to I-E^(k).

Following in vitro challenge of a T cell clone with this analogue, noproliferation was detected although IL-4 secretion was maintained, aswas the capacity of the clone to help B cell responses. In a subsequentstudy the same group demonstrated the separation of T cell-mediatedcytolysis from cytokine production. In this instance, the formerremained unaltered while the latter was impaired. The efficacy ofaltered peptide ligands in vivo was initially demonstrated in a murinemodel of EAE (experimental allergic encephalomyelitis) by McDevitt andcolleagues (Smilek et al (1991) Proc Natl Acad Sci USA 88: 9633-9637).In this model EAE is induced by immunisation with the encephalitogenicpeptide Ac1-11 of MBP (myelin basic protein). Substitution at positionfour (lysine) with an alanine residue generated a peptide which boundwell to its restricting element (Aα^(u)Aβ^(u)), but which wasnon-immunogenic in the susceptible PL/JxSJLF1 strain and which,furthermore prevented the onset of EAE when administered either beforeor after immunisation with the encephalitogenic peptide. Thus, residuescan be identified in peptides which affect the ability of the peptidesto induce various functions of T-cells.

Advantageously, peptides may be designed to favour T-cell proliferationand induction of desensitisation. Metzler and Wraith have demonstratedimproved tolerogenic capacity of peptides in which substitutionsincreasing peptide-MHC affinity have been made (Metzler & Wraith (1993)Int Immunol˜: 1159-65). That an altered peptide ligand can causelong-term and profound anergy in cloned T cells was demonstrated bySloan-Lancaster et al (1993) Nature 363: 156-9.

The compositions of the invention are capable of inducing a late phaseresponse in an individual that is sensitised to the allergens. The term“late phase response” includes the meaning as set forth in Allergy andAllergic Diseases (1997) A. B. Kay (Ed.), Blackwell Science, pp1113-1130. The late phase response may be any late phase response (LPR).Preferably, the peptides are capable of inducing a late asthmaticresponse (LAR) or a late rhinitic response, or a late phase skinresponse or a late phase ocular response. Whether or not a particularpeptide can give rise to a LPR can be determined using methods wellknown in the art; a particularly preferred method is that described inCromwell O, Durham S R, Shaw R J, Mackay J and Kay A B. Provocationtests and measurements of mediators from mast cells and basophils inasthma and allergic rhinitis. In: Handbook of Experimental Immunology(4) Chapter 127, Editor: Weir D M, Blackwell Scientific Publications,1986.

Thus, preferably, the individual peptides and variants of the inventionare able to induce a LPR in an individual who has been sensitised to theallergens. Whether or not an individual has been sensitised to theallergens may be determined by well known procedures such as skin pricktesting with solutions of allergen extracts, induction of cutaneousLPRs, clinical history, allergen challenge and radioallergosorbent test(RAST) for measurement of allergen specific IgE. Whether or not aparticular individual is expected to benefit from treatment may bedetermined by the physician based, for example, on such tests.

Desensitising or tolerising an individual to the allergens meansinhibition or dampening of allergic tissue reactions induced by theallergens in appropriately sensitised individuals. It has been shownthat T cells can be selectively activated, and then renderedunresponsive. Moreover the anergising or elimination of these T-cellsleads to desensitisation of the patient for a particular allergen. Thedesensitisation manifests itself as a reduction in response to anallergen or allergen-derived peptide, or preferably an elimination ofsuch a response, on second and further administrations of the allergenor allergen-derived peptide. The second administration may be made aftera suitable period of time has elapsed to allow desensitisation to occur;this is preferably any period between one day and several weeks. Aninterval of around two weeks is preferred.

Although the compositions of the invention are able to induce a LPR in abirch allergic individual, it should be appreciated that when acomposition is used to treat a patient it is preferable that asufficiently low concentration of the composition is used such that noobservable LPR will occur but the response will be sufficient topartially desensitise the T cells such that the next (preferably higher)dose may be given, and so on. In this way the dose is built up to givefull desensitisation but often without ever inducing a LPR in thepatient. Although, the composition or peptide is able to do so at ahigher concentration than is administered.

The compositions of the invention preferably are capable of inducing alate phase response in 50% or more of a panel of birch allergicindividuals from the population. More preferably, the compositions arecapable of inducing a LPR in 55% or more, 60% or more, 65% or more, 70%or more, 75% or more, 80% or more, 85% or more, or 90% or more ofsensitized individuals in a panel. Whether or not the compositions areable to induce a LPR in a certain percentage of a panel of subjects canbe determined by methods which are well known in the art.

Nucleic Acids and Vectors

The individual peptides that make up the compositions and products ofthe invention may be administered directly, or may be administeredindirectly by expression from an encoding sequence. For example, apolynucleotide may be provided that encodes a peptide of the invention,such as any of the peptides described above. A peptide of the inventionmay thus be produced from or delivered in the form of a polynucleotidewhich encodes, and is capable of expressing, it. Any reference herein tothe use, delivery or administration of a peptide of the invention isintended to include the indirect use, delivery or administration of sucha peptide via expression from a polynucleotide that encodes it.

Accordingly, the invention provides a composition for use in preventingor treating allergy to birch by tolerisation comprising at least onepolynucleotide sequence which when expressed causes the production of acomposition according to the invention for use in preventing or treatingallergy to birch by tolerisation.

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably herein and refer to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Non-limiting examples of polynucleotides include a gene, a genefragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide of theinvention may be provided in isolated or purified form. A nucleic acidsequence which “encodes” a selected polypeptide is a nucleic acidmolecule which is transcribed (in the case of DNA) and translated (inthe case of mRNA) into a polypeptide in vivo when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Forthe purposes of the invention, such nucleic acid sequences can include,but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic sequences from viral or prokaryotic DNA or RNA, and evensynthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence.

Polynucleotides of the invention can be synthesised according to methodswell known in the art, as described by way of example in Sambrook et al(1989, Molecular Cloning—a laboratory manual; Cold Spring Harbor Press).

The polynucleotide molecules of the present invention may be provided inthe form of an expression cassette which includes control sequencesoperably linked to the inserted sequence, thus allowing for expressionof the peptide of the invention in vivo in a targeted subject. Theseexpression cassettes, in turn, are typically provided within vectors(e.g., plasmids or recombinant viral vectors) which are suitable for useas reagents for nucleic acid immunization. Such an expression cassettemay be administered directly to a host subject. Alternatively, a vectorcomprising a polynucleotide of the invention may be administered to ahost subject. Preferably the polynucleotide is prepared and/oradministered using a genetic vector. A suitable vector may be any vectorwhich is capable of carrying a sufficient amount of genetic information,and allowing expression of a peptide of the invention.

Expression vectors are routinely constructed in the art of molecularbiology and may for example involve the use of plasmid DNA andappropriate initiators, promoters, enhancers and other elements, such asfor example polyadenylation signals which may be necessary, and whichare positioned in the correct orientation, in order to allow forexpression of a peptide of the invention. Other suitable vectors wouldbe apparent to persons skilled in the art. By way of further example inthis regard we refer to Sambrook et al.

Thus, a polypeptide of the invention may be provided by delivering sucha vector to a cell and allowing transcription from the vector to occur.Thus, the invention also provides a vector for use in preventing ortreating allergy to birch pollen by tolerisation comprising four or morepolynucleotide sequences which encode a different polypeptide of theinvention. Preferably, a polynucleotide of the invention or for use inthe invention in a vector is operably linked to a control sequence whichis capable of providing for the expression of the coding sequence by thehost cell, i.e. the vector is an expression vector.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given regulatory sequence, such as a promoter,operably linked to a nucleic acid sequence is capable of effecting theexpression of that sequence when the proper enzymes are present. Thepromoter need not be contiguous with the sequence, so long as itfunctions to direct the expression thereof. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween the promoter sequence and the nucleic acid sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

A number of expression systems have been described in the art, each ofwhich typically consists of a vector containing a gene or nucleotidesequence of interest operably linked to expression control sequences.These control sequences include transcriptional promoter sequences andtranscriptional start and termination sequences. The vectors of theinvention may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter. A“plasmid” is a vector in the form of an extrachromosomal geneticelement. The vectors may contain one or more selectable marker genes,for example an ampicillin resistance gene in the case of a bacterialplasmid or a resistance gene for a fungal vector. Vectors may be used invitro, for example for the production of DNA or RNA or used to transfector transform a host cell, for example, a mammalian host cell. Thevectors may also be adapted to be used in vivo, for example to allow invivo expression of the polypeptide.

A “promoter” is a nucleotide sequence which initiates and regulatestranscription of a polypeptide-encoding polynucleotide. Promoters caninclude inducible promoters (where expression of a polynucleotidesequence operably linked to the promoter is induced by an analyte,cofactor, regulatory protein, etc.), repressible promoters (whereexpression of a polynucleotide sequence operably linked to the promoteris repressed by an analyte, cofactor, regulatory protein, etc.), andconstitutive promoters. It is intended that the term “promoter” or“control element” includes full-length promoter regions and functional(e.g., controls transcription or translation) segments of these regions.

A polynucleotide, expression cassette or vector according to the presentinvention may additionally comprise a signal peptide sequence. Thesignal peptide sequence is generally inserted in operable linkage withthe promoter such that the signal peptide is expressed and facilitatessecretion of a polypeptide encoded by coding sequence also in operablelinkage with the promoter.

Typically a signal peptide sequence encodes a peptide of 10 to 30 aminoacids for example 15 to 20 amino acids. Often the amino acids arepredominantly hydrophobic. In a typical situation, a signal peptidetargets a growing polypeptide chain bearing the signal peptide to theendoplasmic reticulum of the expressing cell. The signal peptide iscleaved off in the endoplasmic reticulum, allowing for secretion of thepolypeptide via the Golgi apparatus. Thus, a peptide of the inventionmay be provided to an individual by expression from cells within theindividual, and secretion from those cells.

Alternatively, polynucleotides of the invention may be expressed in asuitable manner to allow presentation of a peptide of the invention byan MHC class II molecule at the surface of an antigen presenting cell.For example, a polynucleotide, expression cassette or vector of theinvention may be targeted to antigen presenting cells, or the expressionof encoded peptide may be preferentially stimulated or induced in suchcells.

In some embodiments, the polynucleotide, expression cassette or vectorwill encode an adjuvant, or an adjuvant will otherwise be provided. Asused herein, the term “adjuvant” refers to any material or compositioncapable of specifically or non-specifically altering, enhancing,directing, redirecting, potentiating or initiating an antigen-specificimmune response.

Polynucleotides of interest may be used in vitro, ex vivo or in vivo inthe production of a peptide of the invention. Such polynucleotides maybe administered or used in the prevention or treatment of allergy bytolerisation.

Methods for gene delivery are known in the art. See, e.g., U.S. Pat.Nos. 5,399,346, 5,580,859 and 5,589,466. The nucleic acid molecule canbe introduced directly into the recipient subject, such as by standardintramuscular or intradermal injection; transdermal particle delivery;inhalation; topically, or by oral, intranasal or mucosal modes ofadministration. The molecule alternatively can be introduced ex vivointo cells that have been removed from a subject. For example, apolynucleotide, expression cassette or vector of the invention may beintroduced into APCs of an individual ex vivo. Cells containing thenucleic acid molecule of interest are re-introduced into the subjectsuch that an immune response can be mounted against the peptide encodedby the nucleic acid molecule. The nucleic acid molecules used in suchimmunization are generally referred to herein as “nucleic acidvaccines.”

The polypeptides, polynucleotides, vectors or cells of the invention maybe present in a substantially isolated form. They may be mixed withcarriers or diluents which will not interfere with their intended useand still be regarded as substantially isolated. They may also be in asubstantially purified form, in which case they will generally compriseat least 90%, e.g. at least 95%, 98% or 99%, of the proteins,polynucleotides, cells or dry mass of the preparation.

Antigen Presenting Cells (APCs)

The invention encompasses the use in vitro of a method of producing apopulation of APCs that present the peptides of the invention on theirsurface, that may be subsequently used in therapy. Such a method may becarried out ex vivo on a sample of cells that have been obtained from apatient. The APCs produced in this way therefore form a pharmaceuticalagent that can be used in the treatment or prevention of birch allergyby tolerisation. The cells should be accepted by the immune system ofthe individual because they derive from that individual. Delivery ofcells that have been produced in this way to the individual from whomthey were originally obtained, thus forms a therapeutic embodiment ofthe invention.

Formulations and Compositions

The peptides, polynucleotides, vectors and cells of the invention may beprovided to an individual either singly or in combination. Each moleculeor cell of the invention may be provided to an individual in anisolated, substantially isolated, purified or substantially purifiedform. For example, a peptide of the invention may be provided to anindividual substantially free from the other peptides. Alternatively,four or more peptides in the composition may be coupled chemicallytogether, using standard peptide coupling reagents, to provide a singlepeptide containing the preferred epitopes. Such peptides would bescreened for basophil histamine release to confirm lack of histaminerelease as per the individual peptides. In a further embodiment, four ormore peptides in the composition may be provided as part of a singlepolypeptide chain i.e by recombinant means from an encodingpolynucleotide. The four or more peptides may be fused contiguously, ormay alternatively be separated by appropriate linkers.

Whilst it may be possible for the peptides, polynucleotides orcompositions according to the invention to be presented in raw form, itis preferable to present them as a pharmaceutical formulation. Thus,according to a further aspect of the invention, the present inventionprovides a pharmaceutical formulation for use in preventing or treatingallergy to birch by tolerisation comprising a composition, vector orproduct according to the invention together with one or morepharmaceutically acceptable carriers or diluents and optionally one ormore other therapeutic ingredients. The carrier (s) must be ‘acceptable’in the sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Typically,carriers for injection, and the final formulation, are sterile andpyrogen free. Preferably, the carrier or diluent is thioglycerol orthioanisole.

Formulation of a composition comprising the peptide, polynucleotides orcells of the invention can be carried out using standard pharmaceuticalformulation chemistries and methodologies all of which are readilyavailable to the reasonably skilled artisan.

For example, compositions containing one or more molecules or cells ofthe invention can be combined with one or more pharmaceuticallyacceptable excipients or vehicles. Auxiliary substances, such as wettingor emulsifying agents, pH buffering substances and the like, may bepresent in the excipient or vehicle. These excipients, vehicles andauxiliary substances are generally pharmaceutical agents that do notinduce an immune response in the individual receiving the composition,and which may be administered without undue toxicity. Pharmaceuticallyacceptable excipients include, but are not limited to, liquids such aswater, saline, polyethyleneglycol, hyaluronic acid, glycerol,thioglycerol and ethanol. Pharmaceutically acceptable salts can also beincluded therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. A thorough discussion of pharmaceuticallyacceptable excipients, vehicles and auxiliary substances is available inRemington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Such compositions may be prepared, packaged, or sold in a form suitablefor bolus administration or for continuous administration. Injectablecompositions may be prepared, packaged, or sold in unit dosage form,such as in ampoules or in multi-dose containers containing apreservative. Compositions include, but are not limited to, suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, andimplantable sustained-release or biodegradable formulations. Suchcompositions may further comprise one or more additional ingredientsincluding, but not limited to, suspending, stabilizing, or dispersingagents. In one embodiment of a composition for parenteraladministration, the active ingredient is provided in dry (for e.g., apowder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration ofthe reconstituted composition. The pharmaceutical compositions may beprepared, packaged, or sold in the form of a sterile injectable aqueousor oily suspension or solution. This suspension or solution may beformulated according to the known art, and may comprise, in addition tothe active ingredient, additional ingredients such as the dispersingagents, wetting agents, or suspending agents described herein. Suchsterile injectable formulations may be prepared using a non-toxicparenterally-acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, butare not limited to, Ringer's solution, isotonic sodium chloridesolution, and fixed oils such as synthetic mono- or di-glycerides.

Other parentally-administrable compositions which are useful includethose which comprise the active ingredient in microcrystalline form, ina liposomal preparation, or as a component of a biodegradable polymersystems. Compositions for sustained release or implantation may comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

Alternatively, the peptides or polynucleotides of the present inventionmay be encapsulated, adsorbed to, or associated with, particulatecarriers. Suitable particulate carriers include those derived frompolymethyl methacrylate polymers, as well as PLG microparticles derivedfrom poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jefferyet al. (1993) Pharm. Res. 10:362-368. Other particulate systems andpolymers can also be used, for example, polymers such as polylysine,polyarginine, polyornithine, spermine, spermidine, as well as conjugatesof these molecules.

The formulation of any of the peptides, polynucleotides or cellsmentioned herein will depend upon factors such as the nature of thesubstance and the method of delivery. Any such substance may beadministered in a variety of dosage forms. It may be administered orally(e.g. as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules), topically, parenterally,subcutaneously, by inhalation, intravenously, intramuscularly,intrasternally, transdermally, intradermally, sublingually,intranasally, buccally or by infusion techniques. The substance may alsobe administered as suppositories. A physician will be able to determinethe required route of administration for each particular individual.

The compositions of formulations of the invention will comprise asuitable concentration of each peptide/polynucleotide/cell to beeffective without causing adverse reaction. Typically, the concentrationof each peptide in the composition will be in the range of 0.03 to 200nmol/ml. More preferably in the range of 0.3 to 200 nmol/ml, 3 to 180nmol/ml, 10 to 150 nmol/ml, 50 to 200 nmol/ml or 30 to 120 nmol/ml. Thecomposition or formulations should have a purity of greater than 95% or98% or a purity of at least 99%.

In one aspect of the invention an adjuvant may be used in combinationwith the polypeptide/polynucleotides/cells of the invention. Theadjuvant is preferably administered in an amount which is sufficient toaugment the effect of the polypeptide/polynucleotides/cells of theinvention or vice versa. The adjuvant or other therapeutic agent may bean agent that potentiates the effects of the molecule of the invention.For example, the other agent may be an immunomodulatory molecule or anadjuvant which enhances the response to the peptide or cell of theinvention.

In one embodiment, therefore, the peptides, polynucleotides, cells orcompositions of the invention are used for therapy in combination withone or more other therapeutic agents. The agents may be administeredseparately, simultaneously or sequentially. They may be administered inthe same or different compositions. Accordingly, in a method of theinvention, the subject may also be treated with a further therapeuticagent.

A composition may therefore be formulated which comprises a moleculeand/or cell of the invention and also one or more other therapeuticmolecules. A composition of the invention may alternatively be usedsimultaneously, sequentially or separately with one or more othertherapeutic compositions as part of a combined treatment.

Non-limiting examples of adjuvants include vitamin D, rapamycin andglucocorticoid steroids such as dexamethasone, fluticasone, budesonide,mometasone, beclomethasone, hydrocortisone, cortisone acetate,prednisone, prednisolone, methylprednisolone, betamethasone andtriamcinolone. A preferred glucocorticoid is dexamethasone.

Therapeutic Methods and Individual to be Treated

The present invention relates to peptides, polynucleotides, vectors andcells that are capable of desensitising or tolerising human individualsto the allergens described above and are therefore useful in theprevention or treatment of birch allergy. The invention providescompositions, products, vectors and formulations for use in preventingor treating allergy to birch by tolerisation. The invention alsoprovides a method of tolerising or desensitizing a birch allergicindividual comprising administering, either singly or in combination thepolypeptides/polynucleotides/cells of the invention as described above.

The individual to be treated or provided with the composition orformulation of the invention is preferably human. It will be appreciatedthat the individual to be treated may be known to be sensitised to theallergens, at risk of being sensitised or suspected of being sensitised.The individual can be tested for sensitisation using techniques wellknown in the art and as described herein. Alternatively, the individualmay have a family history of allergy to birch. It may not be necessaryto test an individual for sensitisation to birch because the individualmay display symptoms of allergy when exposed to birch. By exposure ismeant proximity to, for example, a birch plant, or a substance orproduct derived from a birch plant, or a substance or product containingor comprising either of the above. The substance or product derived froma birch plant is typically birch pollen. By proximity is meant 10 metresor less, 5 metres or less, 2 metres or less, 1 metre or less, or 0metres from the items described above. Symptoms of allergy can includeitchy eyes, runny nose, breathing difficulties, red itchy skin or rash.

The individual to be treated may be of any age. However, preferably, theindividual may be in the age group of 1 to 90, 5 to 60, 10 to 40, ormore preferably 18 to 35.

Preferably, the individual to be treated is from a population that hasMHC allele frequencies within the range of frequencies that arerepresentative of the Caucasian population. Reference population allelefrequencies for 11 common DRB1 allele families are shown in Table 1(Data from HLA Facts Book, Parham and Barber).

TABLE 1 DRB1 1 3 4 7 8 11 12 13 14 15 16 % 6.4 14.7 15.7 8.8 3.4 8.3 3.914.7 2.9 17.6 2.5 Reference 9.4 11.1 12.8 13.2 3.7 13.4 2.3 10.2 3.210.7 3.6 population %Reference frequencies were obtained by analysis of multiple studiesreporting frequencies and the figures shown are mean values. Preferablytherefore, the individual to be treated is from a population that hasequivalent MHC allele frequencies as the reference population for thealleles referred to Table 1 (such as for at least 1, 2, 3, 4, 5 or allof the alleles), for example within the ranges of those figures plus orminus 1, 2, 3, 5, 10, 15 or 20%.

Preferably the individual is from a population where the allelefrequencies of the following DRB1 alleles is:

4—at least 9%7—at least 10%11—at least 8%.

The individual may have had allergy to birch for at least 2 weeks, 1month, 6 months, 1 year or 5 years. The individual may suffer from arash, nasal congestion, nasal discharge and/or coughing caused by theallergy. The individual may or may not have been administered with othercompositions/compounds which treat birch allergy. The individual maylive in a geographical region which has:

a temperate, boreal or arctic climate, and/or:

a typical soil pH in the range of about 3.5, to about 7.5.

The individual typically suffers from allergy to birch pollen in aparticular season. The season typically corresponds to the floweringseason of birch, which is typically spring, preferably early spring (forexample from April to May in the Northern hemisphere). The allergicindividual is typically allergic to birch pollen from any tree in thesubgenus Betula, for example Betula pendula or Betula pubescens.

Combination Immunotherapy

Since many individuals are allergic, or may require desensitizing toseveral polypeptide antigens, the current invention also provides meansof desensitizing individuals that are allergic to multiple antigens.“Tolerance” induced in an individual to a first polypeptide antigen orallergen can create in the individual a “tolergeneic environment”wherein inappropriate immune responses to other antigens can bedownregulated in order to provide tolerance to other antigens.

This finding means that individuals allergic to multiple allergens canbe treated in a greatly reduced time period, and that individualsseriously allergic to some allergens (e.g., peanuts) but more mildlyallergic to other allergens (e.g., cat dander) can benefit from atherapy wherein tolerance to the milder allergen is established and thenthis tolergeneic environment is used to provide tolerance to the other,more extreme allergen. In addition, individuals suffering from anautoimmune disorder who are additionally sensitised (or otherwiseimmune) to an unrelated antigen or allergen can benefit from a treatmentregime wherein tolerance to the unrelated antigen or allergen is firstestablished and then this tolergeneic environment is used to providetolerance to the autoantigen associated with the autoimmune disorder.

A method is therefore provided for desensitising a birch allergicindividual to birch allergen as described above and one or more furtherdifferent polypeptide antigens. The method entails, in a first step,administering to the individual a composition/product/formulation(primary composition) according to the invention as described herein andwherein the administration is carried out in a manner sufficient togenerate a hyporesponsive state against birch allergen. Once ahyporesponsive state has been established toward birch allergen, or atleast a shift toward desensitisation has occurred, the method entailsadministration of a secondary composition comprising a second, differentpolypeptide antigen to which the individual is to be sensitised.Administration of the secondary composition is carried out in such a wayas to take advantage of the tolerogenic environment established by useof the primary composition, where it is now possible to establishtolerance to the second, different polypeptide antigen. The secondarycomposition is coadministered with either the first primary compositionor a larger fragment of the birch allergen. By “coadministered” it ismeant either the simultaneous or concurrent administration, e.g., whenthe two are present in the same composition or administered in separatecompositions at nearly the same time but at different sites, as well asthe delivery of polypeptide antigens in separate compositions atdifferent times. For example, the secondary composition may be deliveredprior to or subsequent to delivery of the first composition at the sameor a different site. The timing between deliveries can range from aboutseveral seconds apart to about several minutes apart, several hoursapart, or even several days apart. Furthermore, different deliverymethods can be employed.

The second polypeptide antigen is preferably an allergen different tothe birch allergen. Suitable allergens for use in the methods of theinvention can of course be obtained and/or produced using known methods.Classes of suitable allergens include, but are not limited to, dust miteallergens, pollens, animal dander (especially cat dander), grassallergens, molds, dusts, antibiotics, stinging insect venoms, and avariety of environmental (including chemicals and metals), drug and foodallergens. Common tree allergens include pollens from cottonwood,popular, ash, birch, maple, oak, elm, hickory, and pecan trees; commonplant allergens include those from mugwort, ragweed, English plantain,sorrel-dock and pigweed; plant contact allergens include those frompoison oak, poison ivy and nettles; common grass allergens include ryegrass, Timothy, Johnson, Bermuda, fescue and bluegrass allergens; commonallergens can also be obtained from molds or fungi such as Alternaria,Fusarium, Hormodendrum, Aspergillus, Micropolyspora, Mucor andthermophilic actinomycetes; epidermal allergens can be obtained fromhouse or organic dusts (typically fungal in origin), or from animalsources such as feathers, and dog dander; common food allergens includemilk and cheese (diary), egg, wheat, nut (e.g., peanut), seafood (e.g.,shellfish), pea, bean and gluten allergens; common environmentalallergens include metals (nickel and gold), chemicals (formaldehyde,trinitrophenol and turpentine), Latex, rubber, fiber (cotton or wool),burlap, hair dye, cosmetic, detergent and perfume allergens; common drugallergens include local anesthetic and salicylate allergens; antibioticallergens include penicillin, tetracycline and sulfonamide allergens;and common insect allergens include bee, wasp and ant venom, andcockroach calyx allergens. Particularly well characterized allergensinclude, but are not limited to, the major cat allergen Fel d1, beevenom phospholipase A2 (PLA) (Akdis et al. (1996) J. Clin. Invest.98:1676-1683) and the multi-epitopic recombinant grass allergen rKBG8.3(Cao et al. (1997) Immunology 90:46-51). These and other suitableallergens are commercially available and/or can be readily prepared asextracts following known techniques.

Preferably, the second polypeptide allergen is a whole tree pollenallergen or allergen fragment selected from the list of allergensequences and database accession numbers (NCBI Entrez accession numbers)below. NCBI is the National Center for Biotechnology information and isa division of the US National Institutes of Health. The NCBI web site,from which access to the database may be sought, iswww.ncbi.nlm.nih.gov/. Allergen sequences and database accession numbers(NCBI Entrez accession numbers):

Olive Tree Olive Sequences

416610 Ole e 1 EDIPQPPVSQFHIQGQVYCDTCRAGFITELSEFIPGASLRLQCKDKENGDVTFTEVGYTRAEGLYSMLVERDHKNEFCEITLISSGRKDCNEIPTEGWAKPSLKFKLNTVNGTTRTVNPLGFFKKEALPKCAQVYNKLGMY PPNM

Tree Allergen Sequences (Mainly Birch) Sequences:

130975 Bet v 2 MSWQTYVDEHLMCDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIMKDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPGQCNMVVERLGDYLIDQGL 1942360 Bet v 2MSWQTYVDEHLMCDIDGQGEELAASAIVGHDGSVWAQSSSFPQFKPQEITGIMKDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPGQCNMVVERLGDYLIDQGL 166953 Bet v 2MSWQTYVDEHLMCDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIMKDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPGQCNMVVERLGDYLIDQGL 541814 Bet v 2MSWQTYVDEHLMCDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIMKDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPGQCNMVVERLGDYLIDQGL 2488678 Bet v 2MSWQTYVDEHLMCDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIMKDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPGQCNMVVERLGDYLIDQGL 1829894 Bet v 2MSWQTYVDEHLMCDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIMKDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPGQCNMVVERLGDYLIDQGL 1168696 Bet v 3MPCSTEAMEKAGHGHASTPRKRSLSNSSFRLRSESLNTLRLRRIFDLFDKNSDGIITVDELSRALNLLGLETDLSELESTVKSFTREGNIGLQFEDFISLHQSLNDSYFAYGGEDEDDNEEDMRKSILSQEEADSFGGFKVFDEDGDGYISARELQMVLGKLGFSEGSEIDRVEKMIVSVDSNRDGRVDFFEFKDM MRSVLVRSS809536 Bet v 4 MADDHPQDKAERERIFKRFDANGDGKISAAELGEALKTLGSITPDEVKHMMAEIDTDGDGFISFQEFTDFGRANRGLLKDVAKIF 543675 Que a I-Quercus alba =oak trees (fragment) GVFTXESQETSVIAPAXLFKALFL543509 Carb I-Carpinus betulus = hornbeam trees (fragment)GVFNYEAETPSVIPAARLFKSYVLDGDKLIPKVAPQAIXK543491 Aln g I-Alnus glutinosa = alder trees (fragment)GVFNYEAETPSVIPAARLFKAFILDGDKLLPKVAPEAVSSVENI 1204056 RubiscoVQCMQVWPPLGLKKFETLSYLPPLSSEQLAKEVDYLLRKNLIPCLEFELEHGFVYREHNRSPGYYDGRYWTMWKLPMFGCNDSSQVLKELEECKKAYP SAFIRIIGFDDK

Additional Tree Allergen Sequences (NCBI Entrez Accession Number):

131919; 128193; 585564; 1942360; 2554672; 2392209; 2414158; 1321728;1321726; 1321724; 1321722; 1321720; 1321718; 1321716; 1321714; 1321712;3015520; 2935416; 464576; 1705843; 1168701; 1168710; 1168709; 1168708;1168707; 1168706; 1168705; 1168704; 1168703; 1168702; 1842188; 2564228;2564226; 2564224; 2564222; 2564220; 2051993; 1813891; 1536889; 534910;534900; 534898; 1340000; 1339998; 2149808; 66207; 2129477; 1076249;1076247; 629480; 481805; 81443; 1361968; 1361967; 1361966; 1361965;1361964; 1361963; 1361962; 1361961; 1361960; 1361959; 320546; 629483;629482; 629481; 541804; 320545; 81444; 541814; 629484; 474911; 452742;1834387; 298737; 298736; 1584322; 1584321; 584320; 1542873; 1542871;1542869; 1542867; 1542865; 1542863; 1542861; 1542859; 1542857; 1483232;1483230; 1483228; 558561; 551640; 488605; 452746; 452744; 452740;452738; 452736; 452734; 452732; 452730; 452728; 450885; 17938; 17927;17925; 17921; 297538; 510951; 289331; 289329; 166953.

Cedar Sequences

493634 Cry j IB precursorMDSPCLVALLVFSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYLAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLYLYGCSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLTSTGVTISNNLFFNHHKVMSLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPHLTQNAGVLTCSLSKRC 493632 Cry j IA precursorMDSPCLVALLVLSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLHLYGCSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLSSTGVTISNNLFFNHHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPQLTKNAGVLTCSLSKRC1076242 Cry j II precursor-Japanese cedarMAMKLIAPMAFLAMQUQLIIMAAAEDQSAQIMLDSVVEKYLRSNRSLRKVEHSRHDAINIFNVEKYGAVGDGKHDCTEAFSTAWQAACKNPSAMLLVPGSKKFVVNNLFFNGPCQPHFTFKVDGIIAAYQNPASWKNNRIWLQFAKLTGFTLMGKGVIDGQGKQWWAGQCKWVNGREICNDRDRPTAIKFDFSTGLIIQGLKLMNSPEFHLVFGNCEGVKIIGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDCVAIGTGSSNIVIEDLICGPGHGISIGSLGRENSRAEVSYVHVNGAKFIDTQNGLRIKTWQGGSGMASHIIYENVEMINSENPILINQFYCTSASACQNQRSAVQIQDVTYKNIRGTSATAAAIQLKCSDSMPCKDIKLSDISLKLTSGKIASCLNDNANGYFSGHVIPACKNLSPSAKRKESKSHKHPKTVMVENMRAYDKGNRTRILLGSRPPNCTNKCHGCSPCKAKLVIVHRIMPQEYYPQRWICSCHGKIYHP 1076241 Cry j II protein-Japanese cedarMAMKFIAPMAFVAMQLIIMAAAEDQSAQIMLDSDIEQYLRSNRSLRKVEHSRHDAINIFNVEKYGAVGDGKHDCTEAFSTAWQAACKKPSAMLLVPGNKKFVVNNLFFNGPCQPHFTFKVDGIIAAYQNPASWKNNRIWLQFAICLTGFTLMGKGVIDGQGKQWWAGQCKWVNGREICNDRDRPTAIKFDFSTGLIIQGLKLMNSPEFHLVFGNCEGVKIIGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDCVAIGTGSSNIVIEDLICGPGHGISIGSLGRENSRAEVSYVHVNGAKFIDTQNGLRIKTWQGGSGMASHIIYENVEMINSENPILINQFYCTSASACQNQRSAVQIQDVTYKNIRGTSATAAAIQLKCSDSMPCKDIKLSDISLKLTSGKIASCLNDNANGYFSGHVIPACKNLSPSAKRKESKSHKHPKTVMVKNMGAYDKGNRTRILLGSRPPNCTNKCHGCSPCKAKLVIVHRIMPQEYYPQRWMCSRHGKIYHP 541803 Cry j I precursor-Japanese cedarMDSPCLVALLVLSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLHLYGCSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLSSTGVTISNNLFFNHHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPQLTKNAGVLTCSLSKRC 541802 Cry j I precursor-Japanese cedarMDSPCLVALLVFSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLYLYGCSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLTSTGVTISNNLFFNHHKVMSLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTTYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPHLTQNAGVLTCSLSKRC

Delivery Methods

Once formulated the compositions of the invention can be delivered to asubject in vivo using a variety of known routes and techniques. Forexample, a composition can be provided as an injectable solution,suspension or emulsion and administered via parenteral, subcutaneous,epidermal, intradermal, intramuscular, intraarterial, intraperitoneal,intravenous injection using a conventional needle and syringe, or usinga liquid jet injection system, or using a patch. Compositions can alsobe administered topically to skin or mucosal tissue, such as nasally,intratracheally, intestinal, rectally or vaginally, or provided as afinely divided spray suitable for respiratory or pulmonaryadministration. Other modes of administration include oraladministration, suppositories, sublingual administration, and active orpassive transdermal delivery techniques.

Where a peptide of the invention is to be administered, it is preferredto administer the peptide to a site in the body where it will have theability to contact suitable antigen presenting cells, and where it, orthey, will have the opportunity to contact T cells of the individual.Where an APC is to be administered, it is preferred to administer theAPC to a site in the body where it will have the ability to contact, andactivate, suitable T cells of the individual.

Delivery Regimes

Administration of the peptides/polynucleotides/cells (such as thecomposition containing a plurality of peptides) may be by any suitablemethod as described above. Suitable amounts of the peptide may bedetermined empirically, but typically are in the range given below. Asingle administration of each peptide may be sufficient to have abeneficial effect for the patient, but it will be appreciated that itmay be beneficial if the peptide is administered more than once, inwhich case typical administration regimes may be, for example, once ortwice a week for 2-4 weeks every 6 months, or once a day for a weekevery four to six months. As will be appreciated, each peptide orpolynucleotide, or combination of peptides and/or polynucleotides may beadministered to a patient singly or in combination.

Dosages for administration will depend upon a number of factorsincluding the nature of the composition, the route of administration andthe schedule and timing of the administration regime. Suitable doses ofa molecule of the invention may be in the order of up to 15 μg, up to 20μg, up to 25 μg, up to 30 μg, up to 50 μg, up to 100 μg, up to 500 μg ormore per administration. Suitable doses may be less than 15 μg, but atleast 1 ng, or at least 2 ng, or at least 5 ng, or at least 50 ng, orleast 100 ng, or at least 500 ng, or at least 1 μg, or at least 10 μg.For some molecules of the invention, the dose used may be higher, forexample, up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg orhigher. Such doses may be provided in a liquid formulation, at aconcentration suitable to allow an appropriate volume for administrationby the selected route.

Kits

The invention also relates to a combination of components describedherein suitable for use in a treatment of the invention which arepackaged in the form of a kit in a container. Such kits may comprise aseries of components to allow for a treatment of the invention. Forexample, a kit may comprise one or more different peptides,polynucleotides and/or cells of the invention, or one or more peptides,polynucleotides or cells of the invention and one or more additionaltherapeutic agents suitable for simultaneous administration, or forsequential or separate administration. The kit may optionally containother suitable reagent(s) or instructions and the like.

The invention is illustrated by the following Examples:

Example 1 MHC Class II Binding Search

The aim of this study is to identify a distinct panel of peptides withstrong affinities for the eight most common human MHC Class II HLA-DRB1*allotypes. In order to identify binding peptides in the major birchallergens Bet v1, Bet v2, Bet v3, Bet v4, Bet v6, an in silico approachwas carried out using the commercially available EpiMatrix algorithm(EpiVax Inc.) This is a bioinformatic analysis of peptides from asequence for the potential to be accommodated within the binding grooveof MHC class II HLA-DR molecules.

EpiMatrix is a matrix-based algorithm that ranks 9 amino acid residuesequences, overlapping by 8 amino acids, from any polypeptide sequenceby estimated probability of binding to each of the selected MHCmolecules. (De Groot et al., AIDS Research and Human Retroviruses13:539-41 (1997). The procedure for developing matrix motifs waspublished by Schafer et al, Vaccine 16:1880-4 (1998). In this Example,binding potential for HLA DR1, DR3, DR4, DR7, DRB, DR11, DR13 and DR15is assessed. Putative MHC ligands are selected by scoring each 9-merframe in a protein sequence. This score is derived by comparing thesequence of the 9-mer to the matrix of amino acid sequences known tobind to each MHC allele. Retrospective studies have demonstrated thatEpiMatrix accurately predicts published MHC ligands (Jesdale et al., inVaccines '97 (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,1997)). Successful Prediction of peptides which bind to multiple MHCmolecules has also been confirmed.

The EpiMatrix data for each allergen is shown either:

As overlapping 9 mer peptide data with the binding (Z) score for eachallele and the number of ‘hits’ for the eight alleles (Z scores of equalto or greater than the top 5% of predicted binders): or

As a cluster report where the data from analysing multiple sequencesfrom the database is ‘clustered’ to give an overview of binding for allvariants of the protein. The “EpiMatrix hits’ refers to the number ofhigh predicted Z binding scores for the eight alleles within thatsequence whilst the “EpiMatrix Cluster Score” is derived from the numberof hits normalized for the length of the cluster. Cluster Score is thusthe excess or shortfall in predicted aggregate MHC binding propertiesrelative to a random peptide standard. A cluster score above 10 isconsidered to indicate broad MHC binding properties.

EpiMatrix analyses were performed on the entire sequences of knownisoforms of Bet v1, listed below with their corresponding NCBI accessionnumbers:

Bet v1 L P43185; Bet v1 M/N P43186; Betv1 K P43184; Bet v1 J P43183; Betv1 G P43180; Bet v1 F/I P43179; Bet v1 E P43178; Bet v1 D/H P43177; Betv1 C P43176; Bet v1 B P45431; Bet v1 A P15494;Epimatrix analyses were also performed on additional known Bet v1sequences indexed by accession number in Table 2.

These analyses identified core peptides (and their flanking sequences)derived from the above sequences which are predicted to have good MHCclass-II binding.

These sequences are shown below in Tables 1 and 2. As shown, many of thepeptides identified are highly conserved between different Bet v1isoforms.

In Tables 1 and 2:

“Residues in main sequence” gives the location of the peptide within thesequences that were analysed. The core peptide (underscored middle aminoacids in bold) defines the actual binding sequence that was identifiedduring the analysis. The stabilizing flanks (N-terminal and C-terminal,not bold) were included for use with the core sequence and are typicallyrequired to aid manufacture of the peptides. “Number of hits” refers tothe number of high predicted binding affinities for all MHC types testedwithin the sequence. The “EpiMatrix Cluster Score” is derived from thenumber of hits normalized for the length of the cluster. Cluster Scoreis thus the excess or shortfall in predicted aggregate MHC bindingproperties relative to a random peptide standard. A score above 10 isconsidered to indicate broad MHC binding properties.

TABLE 1 Bet v1 EpiMatrix EpiMatrix CLUSTER INPUT RESIDUES IN Hydro- HITSSCORE Peptide SEQ SE- SEQUENCE pho- (Excl (Excl ID ID QUENCE(Incl. FLANKS) SEQUENCE bicity FLANKS) FLANKS) NO NO: P43185 13-28VIPAARMFKAFILDGD 0.78 6 11.3 P1 1 P15494 100-114 SNEIKIVATPDGGSI 0.03 715.57 P2 2 P43176 100-114 CNEIKIVATPDGGSI 0.25 7 15.57 P3 3 P43177100-114 SNEIKIVATPDGGCV 0.23 7 15.57 P4 4 P43178 100-116SNEIKIVATPNGGSILK 0.02 10 20.13 P5 5 P43179 100-116 SNEIKIVATPNGGSILK0.02 10 20.13 P6 P43180 100-114 SNEIKIVATPDGGCV 0.23 7 15.57 P7 P43183100-116 SNEIKIVATPNGGSILK 0.02 10 20.13 P8 P43184 100-114CNEIKIVATPDGGSI 0.25 7 15.57 P9 P43185 100-114 SNEIKIVATPDGGCV 0.23 715.57 P10 P43186 100-114 CNEIKIVATPDGGSI 0.25 7 15.57 P11 P45431 100-114CNEIKIVATPDGGSI 0.25 7 15.57 P12 P43178 112-126 GSILKINNKYHTKGD −1.08 612.34 P13 P43179 112-126 GSILKINNKYHTKGD −1.08 6 12.34 P14 P43183112-126 GSILKIHNKYHTKGD −1.08 6 12.34 P15 P15494 142-160ETLLRAVESYLLAHSDAYN −0.09 8 12.06 P16 6 P43176 142-160EALLRAVESYLLAHSDAYN 0.04 8 12.06 P17 7 P43177 142-160ETLLRAVESYLLAHSDAYN −0.09 8 12.06 P18 P43178 142-160 ETLLRAVESYLLAHSDAYN−0.09 8 12.06 P19 P43179 142-160 ETLLRAVESYLLAHSDAYN −0.09 8 12.06 P20P43180 142-160 ETLLRAVESYLLAHSDAYN −0.09 8 12.06 P21 P43183 142-160ETLLRAVESYLLAHSDAYN −0.09 8 12.06 P22 P43184 142-160 ETLLRAVESYLLAHSDAYN0.04 8 12.06 P23 P43185 142-160 ETLLRAVESYLLAHSDAYN −0.09 8 12.06 P24P43186 142-160 ETLLRAVESYLLAHSDAYN 0.04 8 12.06 P25 P45431 142-160ETLLRAVESYLLAHSDAYN 0.04 8 12.06 P26

TABLE 1A EpiMatrix analysis of Bet v1 Sequence: P15494: predictedmultiple HLA DR allele binding region: FNYETETTSVIPAARLFKAFILDGDNLF (4-31).DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* Frame AA Frame 0101 03010401 0701 0801 1101 1301 1501 Start Sequence Stop Z−Score Z−ScoreZ−Score Z−Score Z−Score Z−Score Z−Score Z−Score Hits  4 FNYETETTS 12 .961.29 1.58 −.42 .10 .93 −.21 .27 0  5 NYETETTSV 13 .32 −.86 .59 .81 −1.32−.80 −.75 .22 0  6 YETETTSVI 14 2.15 1.37 2.25 1.72 1.09 1.15 −.03 .70 3 7 ETETTSVIP 15 −.11 −1.34 .48 .58 −.89 −.60 −.84 −.03 0  8 TETTSVIPA 16.16 −1.10 .97 .51 −.86 −.69 −.48 .04 0  9 ETTSVIPAA 17 −.04 .16 .64 −.18−.89 .43 −.39 −.17 0 10 TTSVIPAAR 18 .64 .66 −.12 −.56 .14 .47 .50 −.430 11 TSVIPAARL 19 1.52 −.44 .23 .47 −.63 .28 −.96 −.33 0 12 SVIPAARLF 20.69 −.06 −.07 1.07 −.44 .25 1.05 −.20 0 13 VIPAARLFK 21 .67 2.00 .65 .501.21 1.32 1.98 1.21 2 14 IPAARLFKA 22 .55 1.17 .00 −.28 .32 .16 1.271.60 0 15 PAARLFKAF 23 −1.29 −1.65 −1.57 .28 −.29 −.88 −.32 −.58 0 16AARLFKAFI 24 1.13 1.39 −.25 −.68 1.25 1.50 1.32 −.06 0 17 ARLFKAFIL 252.12 .89 .25 1.84 1.11 .18 1.54 3 18 RLFKAFILD 26 −1.54 −.90 −1.35 −.861.38 −1.29 −.38 −.67 0 19 LFKAFILDG 27 .86 1.13 1.48 .68 .94 .39 1.141.37 0 20 FKAFILDGD 28 −.17 .58 .09 −.43 1.90 .24 .30 .96 1 21 KAFILDGDN29 −.49 −1.35 −.67 −.69 −.59 −.02 −2.05 .29 0 22 AFILDGDNL 30 1.32 −.28−.21 .67 −.85 −.69 −.80 .30 0 23 FILDGDNLF 31 −.02 2.27 1.73 1.07 .47−.09 −.22 .74 2

TABLE 2 Bet v 1 EpiMatrix EpiMatrix CLUSTER INPUT RESIDUES IN Hydro-HITS SCORE Peptide SEQ SE- SEQUENCE pho- (Excl (Excl ID ID QUENCE(Incl. FLANKS) SEQUENCE bicity FLANKS) FLANKS) NO NO: CAA04829  1-16GVFNYEIGATSVIPAA 0.84 7 11.23 P27 8 2122374C 14-34 IAPARLFRSFVLDADNLIPKV0.62 9 13.67 P28 9 ABC41588  97-111 CNEIKLVATPDGGST -0.15 7 15.45 P29 10ABC41605  97-111 SKEIKIAAAPDGGSI 0.01 6 13.05 P30 11 ABC41615  97-111SNEIKIVATPDGGCI 0.25 7 15.57 P31 12 ABC41617  97-1ll CNEIKLVATPDGGSI0.20 7 15.45 P32 13 ABC41596  97-113 CNEIKIVAAPGGGSILK 0.54 9 17.42 P3314 ABC41602  97-113 CNEIKIVPAPGGGSILK 0.34 9 16.98 P34 15 ABC41609 97-113 SYEIKIVAAPGGGSILK 0.48 9 17.42 P35 16 1QMR_A  99-113SNEIKIVATGDGGSI 0.11 5 10.32 P36 17 CAA96546 100-114 CNEIKIVAAPDGGSI0.41 7 14.59 P37 18 CAA96547 100-114 SNEIKIVATPDGRSI -0.25 7 15.57 P3819 CAA07324 100-114 SNEIKLVATPDGGSI -0.02 7 15.45 P39 20 CAA07327100-114 CNEIKIVATPDGGCV 0.45 7 15.57 P40 21 CAA07318 100-114SNEIKIVTTPDGGCV 0.06 7 15.57 P41 22 AAD26561 100-114 SNEIKIVATPDGGPI-0.03 7 15.57 P42 23 ABC41589 109-125 GSILKIRNKYHTKGDHE -1.41 9 15.34P43 24 ABC41609 139-150 AGLFKAVENYLV 0.82 5 10.95 P44 25 ABC41583139-150 ETLLRAVESYLL 0.58 6 13.44 P45 26 ABC41589 139-150 EALLRAVESYLL0.78 6 13.44 P46 27 ABC41602 139-150 EALFRAVESYLL 0.70 7 16.84 P47 28CAA96544 139-156 EKAVGLLKAVESYLLAHS 0.43 7 12.67 P48 29 CAA07319 141-160GETLLRAVEGYLLAHSDAYN -0.09 8 11.13 P49 30 AAD26561 142-156ETLLRAVESYPLAHS -0.05 6 13.44 P50 31 2122374C 142-160AGLFKAVENYLVAHPNAYN 0.02 10 15.5 P51 32 CAA96539 142-160ETLLRAVERYLLAHSDAYN -0.29 10 18.69 P52 33 2122374A 142-160ETLLRAVESYLLAHSDAYN -0.02 9 15.46 P53 34

Example 2

EpiMatrix analyses as above were performed on the entire sequence of aknown isoform of Bet v 3 (NCBI accession no: P43187). These analysesidentified a core peptide (and its flanking sequence) derived from theabove sequence which is predicted to have good MHC class-II binding. Thesequence is shown below in Table 3. Headings and notes for Table 3 areas with Table 1 above.

TABLE 3 Bet v 3 EpiMatrix EpiMatrix CLUSTER INPUT RESIDUES IN Hydro-HITS SCORE Peptide SEQ SE- SEQUENCE pho- (Excl (Excl ID ID QUENCE(Incl. FLANKS) SEQUENCE bicity FLANKS) FLANKS) NO NO: P43187 188-205VDFFEFKDMMRSVLVRSS 0.16 10 14.02 P54 35A sequence at residues 80 to 94 of P43187, TVKSFTREGNIGLQF (Peptide IDNO. P55, SEQ ID NO: 36), was also predicted to have good MHC-Class IIbinding. Additional in silico analysis of other birch allergen sequencesfrom Bet v 3 is shown here:

TABLE 3A EpiMatrix analysis of Bet v3 Sequence: GI1168696_SPP43187:predicted multiple HLA DR allele binding region SLNTLRLRRIFDLFDK (35-50).DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* Frame 0101 0301 04010701 0801 1101 1301 1501 start sequence end Z-Score Z-Score Z-ScoreZ-Score Z-Score Z-Score Z-Score Z-Score Hits 35 SLNTLRLRR 43 .28 .97-.38 .38 .64 .53 1.73 .68 1 36 LNTLRLRRI 44 1.30 .87 -.15 .46 .82 1.351.41 .17 0 37 NTLRLRRIF 45 -.24 .12 -1.52 .43 1.21 1.13 1.42 -.47 0 38TLRLRRIFD 46 .47 .40 -.46 .42 .66 .49 -.34 1 39 LRLRRIFDL 47 1.09 .83.08 1.68 .31 1.94 4 40 RLRRIFDLF 48 -1.83 -.86 -1.40 -.38 -.94 -1.34-.63 -.87 0 41 LRRIFDLFD 49 .84 .60 1.39 .46 1.41 .88 .39 1.55 0 42RRIFDLFDK 50 -.31 .38 -.31 -.58 .85 .33 1.05 1.61 0

Example 3

EpiMatrix analyses as above were performed on the entire sequence ofknown isoforms of Bet v 4 (NCBI accession nos: Q39419, CAA73147). Theseanalyses identified core peptides (and their flanking sequences) derivedfrom the above sequences which are predicted to have good MHC class-IIbinding. These sequences are shown below in Table 4. Headings and notesfor Table 4 are as with Table 1 above.

TABLE 4 Bet v 4 EpiMatrix EpiMatrix CLUSTER INPUT RESIDUES IN Hydro-HITS SCORE Peptide SEQ SE- SEQUENCE pho- (Excl (Excl ID ID QUENCE(Incl. FLANKS) SEQUENCE bicity FLANKS) FLANKS) NO NO: Q39419 10-27AERERIFKRFDANGDGKI -1.19 8 13.83 P56 37 Q39419 67-81 FTDFGRANRGLLKDV-0.38 7 13.49 P57 38 CAA73147 67-81 FTDFARANRGLLKDV -0.23 7 13.53 P58 39

Example 4

EpiMatrix analyses as above were performed on the entire sequence of aknown isoform of Bet v 6 (NCBI accession no: 065002). These analysesidentified core peptides (and their flanking sequences) derived from theabove sequences which are predicted to have good MHC class-II binding.These sequences are shown below in Table 5. Headings and notes for Table5 are as with Table 1 above.

TABLE 5 Bet v 6 EpiMatrix EpiMatrix CLUSTER INPUT RESIDUES IN Hydro-HITS SCORE Peptide SEQ SE- SEQUENCE pho- (Excl (Excl ID ID QUENCE(Incl. FLANKS) SEQUENCE bicity FLANKS) FLANKS) NO NO: 065002 43-63PVKGEKLVEKFKGLGVTLLHGD 0.04 9 14.09 P59 40 065002 67-90HESLVKAFKQVDVVISTVGHLQ 0.52 11 15.54 P60 41 LA 065002 149-170YVSSNFFAGYFLPTLAQPGLTS 0.46 10 12.92 P61 42 065002 258-274PINVILAINHSVFVKGD 0.83 7 10.54 P62 43

Example 5

EpiMatrix analyses as above were performed on the entire sequence of aknown isoform of Bet v 7 (NCBI accession no: CAC84116). These analysesidentified a core peptide (and its flanking sequence) derived from theabove sequence which is predicted to have good MI-IC class-H binding.This sequence is shown below in Table 6. Headings and notes for Table 6are as with Table 1 above.

TABLE 6 Bet v 7 EpiMatrix EpiMatrix CLUSTER INPUT RESIDUES IN Hydro-HITS SCORE Peptide SEQ SE- SEQUENCE pho- (Excl (Excl ID ID QUENCE(Incl. FLANKS) SEQUENCE bicity FLANKS) FLANKS) NO NO: CAC84116 34-48AENFRALCTGEKGNG −0.77 5 10.46 P63 44

Example 5A

Additional in silico analysis of other birch allergen sequences from Betv 2 is shown here:

TABLE 6A EpiMatrix analysis of Bet v2 Sequence: GI1942360_PDB1CQA:predicted multiple HLA DR allele binding region: SVWAQSSSFPQFKPQEITGIMK (33-54).DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* Frame 0101 0301 04010701 0801 1101 1301 1501 start sequence end Z-Score Z-Score Z-ScoreZ-Score Z-Score Z-Score Z-Score Z-Score Hits 33 SVWAQSSSF 41 .75 .401.04 1.19 −.29 −.22 −.17 .71 0 34 VWAQSSSFP 42 .90 .82 1.31 .46 −.02 .21.64 1.01 0 35 WAQSSSFPQ 43 1.70 .36 2.24 1.23 1.44 .12 1.53 3 36AQSSSFPQF 44 −.65 .38 −.28 −.01 −1.26 −.18 −.18 −.56 0 37 QSSSFPQFK 45−.31 .01 .24 .04 −1.01 .00 −1.52 −.51 0 38 SSSFPQFKP 46 −.45 −.73 −1.50−1.02 −.04 −.76 −.02 .21 0 39 SSFPQFKPQ 47 −1.10 −2.37 −.57 .24 −.52−.64 −1.22 −.98 0 40 SFPQFKPQE 48 −.23 1.11 −.75 −1.69 1.34 .47 1.32−.32 0 41 FPQFKPQEI 49 1.55 .73 .67 .99 .88 .49 .09 2.02 1 42 PQFKPQEIT50 −.26 .27 −.85 .12 1.00 .28 .59 −.60 0 43 QFKPQEITG 51 −1.28 −.42 −.84−.60 −.27 −1.13 .23 .00 0 44 FKPQEITGI 52 1.94 1.68 1.69 1.76 .07 .451.37 1.43 4 45 KPQEITGIM 53 .15 .15 .32 .12 −.39 .10 −1.15 −.26 0 46PQEITGIMK 54 −.06 −1.11 −.06 −.24 −1.55 −.85 −1.48 −.15 0

TABLE 6BEpiMatrix analysis of Bet v2 Sequence: GI1942360_PDB1CQA: predictedmultiple HLA DR allele binding region: IKYMVIQGEAGAVIRGKKGSGG (72-93).DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* Frame 0101 0301 04010701 0801 1101 1301 1501 start sequence end Z-Score Z-Score Z-ScoreZ-Score Z-Score Z-Score Z-Score Z-Score Hits 72 IKYMVIQGE 80 1.21 1.601.25 .49 1.87 1.30 −.52 .97 1 73 KYMVIQGEA 81 .45 .63 −.35 .37 .56 1.73.28 .53 1 74 YMVIQGEAG 82 1.68 .22 1.47 .98 .61 1.06 −.37 .26 1 75MVIQGEAGA 83 1.49 2.23 1.51 −.42 1.07 1.87 2.27 .85 3 76 VIQGEAGAV 841.12 1.46 −.18 1.12 −.11 .16 −.07 2.02 1 77 IQGEAGAVI 85 1.64 1.28 .54.46 .67 .50 .86 1 78 QGEAGAVIR 86 .56 .61 −.43 −.01 −.68 −1.08 .29 .12 079 GEAGAVIRG 87 .02 .36 .41 −.45 −.52 −1.38 −.79 .28 0 80 EAGAVIRGK 88−.12 −.55 .17 −.08 −.44 .08 −.23 −1.21 0 81 AGAVIRGKK 89 −.65 .95 −1.14.00 .75 1.71 .58 .53 1 82 GAVIRGKKG 90 .82 −1.39 −.20 −.20 −.70 −.26−1.31 .08 0 83 AVIRGKKGS 91 −.08 .16 −.55 −.21 1.97 1.49 1.43 1.07 1 84VIRGKKGSG 92 .10 −.64 −.90 1.35 1.18 .92 1.79 2 85 IRGKKGSGG 93 1.181.05 .64 .02 2.09 .95 1.11 1.83 2

Example 6

Based on the analyses performed in Examples 1 to 5A, the followingpeptides shown in Table 7 were designed for screening in subsequentassays. The design process involved modification of native sequences toenhance solubility and other physicochemical characteristics. Forexample, for Bir12A, residues in parent 62-77R indicates that thepeptide sequence of Bir12A corresponds to residues 62 to 77 of theparent sequence, with an additional R residue added to the C terminus toimprove solubility. Similarly, for Bir01F, G, H and I, residues inparent 4-18K indicates that these peptide sequences correspond toresidues 4 to 18 of the parent sequence, with an additional K residueadded to the C terminus to improve solubility.

TABLE 7 Peptide Sequence Residues SEQ. ID. NO BIR01F FNYETEATSVIPAARK4-18K (P43185) Bet v1 45 BIR01G FNYEIEATSVIPAARK 4-18K (P43179) Bet v146 BIR01H FNYEIETTSVIPAARK 4-18K (P43177) Bet v1 47 BIR01IFNYETETTSVIPAARK 4-18K (P15494) Bet v1 48 BIR02D PAARMFKAFILDGDKLVPK15-33 (P43185) Bet v1 49 BIR02E PAARLFKAFILEGDTLIPK15-33 (P43184) Bet v1 50 BIR02G PAARLFKAFILEGDNLIPK15-33 (P41380) Bet v1 51 BIR02I PAARMFKAFILD 15-26 (P41385) Bet v1 52BIR02J PAARMFKAFILEGDKLVPK D to E variant of 53 BIR02D BIR04PGTIKKISFPEGFPFKYV 51-68 (P43185) Bet v1 54 BIR05 SPFKYVKERVDEVDHA63-78 (P43186) Bet v1 55 BIR05A FPFKYVKDRVDEVDHT 63-78 (P43185) Bet v156 BIR06 ANFKYSYSMIEGGALGD 78-94 (P43186) Bet v1 57 BIR06BTNFKYSYSVIEGGPVGD 78-94 (P43183) Bet v1 58 BIR06D TNFKYNYSVIEGGPIG78-93 (P) Bet v1 59 BIR07 SNEIKIVATPDGGSILK 100-116 Bet v1 60 BIR07ASNEIKIVATPNGGSILK 100-116 Bet v1 61 BIR07B SNEIKIVATPQGGSILK100-116 Bet v1 62 BIR07C SNEIKIVATPEGGSILK 100-116 Bet v1 63 BIR07DSNEIKIVATPGGGSILK 100-116 Bet v1 64 BIR08 GSILKINNKYHTKGD 112-126 Bet v165 BIR08A SILKISNKYHTKGD 113-125 (P43186) Bet v1 66 BIR09ETLLRAVESYLLAHSDAY 142-159 Bet v1 67 BIR09A GETLLPAVESYLLAHS141-156 Bet v1 68 BIR09B KEMGETLLRAVESYLLAHS 138-156 Bet v1 69 BIR09CKEKGETLLRAVESYLLAHS M to K variant of above 70 BIR10 GSVWAQSSSFPQFK33-45 (P25816) Bet v2 71 BIR11 FPQFKPQEITGIMK 41-54 (AAB44348) Bet v2 72BIR12A PTGMFVAGAKYMVIQGR 62-77R (P35079) Phl p12 73 BIR12BAKYMVIQGEPGRVIRGK 70-86 (P35079) Phl p12 74 BIR13 GIKYMVIQGEAGAVIRGK71-88 (AAB44348) Bet v2 75 BIR14 EAGAVIRGKKGSGGIT 80-95 (P25816) Bet v276 BIR15 SLNTLRLRRIFDLFDK 35-50 Bet v3 77 BIR16A AERERIFKRFDANGEGK10-26 D to E variant 78 Bet v4 BIR16B AERERIFKRFDAGGEGKN to G variant of above 79 BIR17 VKGKLVEKFKGLGVTLLHG 44-62 Bet v6 80

Example 7 In Vitro Binding Analysis

The peptides identified as being potential MHC Class II-binding arepre-screened for solubility in an aqueous, acidic milieu and thepeptides are tested in an in vitro MHC Class II binding assay.

Methods

The assay employed is a competitive MHC class II binding assay, whereineach peptide is analysed for its ability to displace a known controlbinder from each of the human MHC class II allotypes investigated. Theallotypes and control peptides used in this study are typically thoseshown below:

Control Peptides Used in the In Vitro Binding Assays

Allotype Control Peptide Sequence DRB1*0301 Myco. tuberculosis/AKTIAYDEEARRGLE leprae hsp 65 2-16 DRB1*1101 Influenza haemagglutininPKYVKQNTLKLAT 307-319 DRB1*1501 Human myelin basic proteinENPVVHFFKNIVTPR 85-99Each of the peptides from Tables 1 to 7 are analysed in the competitionassay and screened for relative binding compared to the controlpeptides. Due to the nature of the competitive assay the data for eachpeptide is determined as a ratio of its own IC50 to that of the controlpeptide. Thus, a peptide that has an IC50 value that is parity to thecontrol peptide has an identical binding affinity, while peptides with aratio less than one have a higher affinity and those with a ratiogreater than one have a lower affinity.

Solubility in aqueous solution is an essential criterion for a peptideto be an effective therapeutic agent. Therefore, as a consequence of thesolubility screen very hydrophobic peptides with a high frequency oflarge hydrophobic amino acid residues in multiple binding registers willbe eliminated. This is a characteristic of promiscuous HLA-DRB1*binders. Peptides which bind to one or more of the MHC Class IIallotypes are identified. It would be expected that such peptides wouldhave the ability to bind similar allotypes that have not been testedthrough the homology of MHC structures.

Example 8

The following methods are applied to the same peptides as in Example 7.

Cell Proliferation Assay

The cell proliferation assay is performed on PBMC's (140×10⁶ cellsrequired for all parameters to be tested). Proliferation is measured bythe incorporation of the radiolabelled compound 3H-thymidine. In moredetail, 100 μl of the appropriate antigen or peptide concentration isdistributed into the appropriate wells of 96 well plates. The plates arethen placed into a humidified 5% CO2 incubator set at 37° C. for amaximum of 4 hours. PBMC's isolated as described above are prepared to aconcentration of 2×10⁶ cells/ml in complete medium at room temperature.100 μl of cell solution is then distributed into each of the wells ofthe 96 well plates containing antigen/peptide. The plates are thenincubated for 6 to 8 days. The cultures are pulsed with tritiatedthymidine solution by adding 10 μl of tritiated thymidine stock solution(1.85 MBq/ml in serum-free RPMI medium) to each well. The plates arethen returned to the incubator for between 8 and 16 hours. Cultures arethen harvested using a Canberra Packard FilterMate 196 cell harvester.Dried filter mats are counted using an appropriate beta scintillationcounter.

Counts from wells containing peptide are compared statistically to wellscontaining media alone (12 wells per group). The non-parametricMann-Whitney test is used. The same statistical test is used for allsubjects. A statistically significant difference between media onlywells and peptide-stimulated wells is considered a positive stimulationof PBMC's by the peptide.

Cytokine Release Assay

The 36 peptides were manufactured at small scale (approximately 10 mgbatch size, non-GMP). The purity of each peptide was at least 95% byHPLC. 96 well culture plates containing peptides and controls (thenegative control was culture medium and the positive controls werestaphylococcal enterotoxin B (SEB) 25 ng/ml and whole birch pollenallergen extract 100 μg/ml) were prepared in advance and stored at −20°C. prior to the day of assay. Peptides were added to wells in a volumeof 100 μl containing peptides at a concentration of 200 μg/ml, such thatsubsequent addition of 100 μl of cells would create a final assayconcentration of 100 μg/ml.

Peripheral blood mononuclear cells (PBMCs) were isolated fromheparinised blood by Ficoll density gradient centrifugation. A 100 μlaliquot of a 5×10⁶ cell/ml PBMC suspension was then added to each welland the plates placed in a humidified 5% CO2 incubator at 37° C. for 5days. Following stimulation, culture supernatants (100 μl) wereharvested for testing by multiplex bead assay.

Multiplex cytokine bead assays (IL-10, IL-13, Interferon gamma (IFN-g))were performed on thawed culture supernatants according to themanufacturer's instructions. Single measurements were performed for eachculture supernatant sample. After completion of the multiplex assay,individual cytokine levels were determined by interpolation from thestandard curve generated in the assay. A positive result was taken asbeing greater than 100 pg/ml for the IL-13 and IFN-g assays or >4 timesthe background for the EL-10 assays. The number of responders out of 47birch allergic subjects tested was calculated for each peptide for thethree cytokines. Results for IL-13 or IFN-g are summarized in Table 9.

TABLE 8 % responders indicates the proportion of subjects in which eachpeptide induced IL-13 or IFN-g above a threshold level of 100 pg/mlPeptide % responders Bir02J 48 Bir01I 42 Bir01F 38 Bir12B 38 Bir01G 33Bir04 33 Bir09 33 Bir02E 31 Bir02G 31 Bir02I 31 Bir07 31 Bir07C 31Bir09A 31 Bir09B 31 Bir11 31 Bir16A 31 Bir02D 29 Bir09C 27 Bir15 27Bir16B 27 Bir01H 25 Bir06D 25 Bir07B 25 Bir07D 25 Bir10 25 Bir14 25Bir17 25 Bir05A 23 Bir06 23 Bir07A 23 Bir13 23 Bir06B 19 Bir08A 19 Bir0517 Bir08 17 Bir12A 17

Peptides which induce positive response in a high proportion of subjectsare desirable for inclusion in a vaccine. As shown, the top performingpeptides were Bir02J (top of the 02 series), Bir01I (top of the 01series) and Bir12B. The core of any vaccine should ideally contain thesepeptides. The second best performing peptides were Bir04 and Bir09 (topof the 09 series) which may be added to the core mixture of Bir02J,Bir01I and Bir12B. The third best performing peptides were Bir07,Bir07C, Bir11 and Bir16A. Additional peptides from this group may beadded to the vaccine mixture to further increase coverage. Bir15 was thefourth best performing peptide and may also be added to the vaccinemixture. In terms of other peptides in the various series, Bir01F, 01Gor 01H, in that order of preference, are useful variants of Bir01I;Bir02E, 02G, 02I or lastly 02D are useful variants of Bir02J; Bir09A,09B or lastly 09C are useful variants of Bir09; and Bir16B is a usefulvariant of Bir16A. A possible preferred mixture would therefore includeBir02J, Bir01I, Bir12B, Bir04, Bir09, Bir07C and Bir16A. Bir11 and/orBir15 may also be included, or alternatively substituted for Bir07Cand/or Bir16A.

In terms of IL-10 release, Bir01I, listed above as one of the top 3peptides for IL-13 or IFN-g production, induced IL-10 responses in 49%of individuals. Bir02I also induced IL-10 production in a highproportion of individuals (43%). Inclusion of a strong IL-10 inducingpeptide may assist in the induction of tolerance following vaccination.

Example 9 Solubility Screening A) Introduction

TABLE 9-1 Birch peptides to be included in solubility testing Theo-retical Iso- electric Mw Length point Peptide Sequence (Da) (a.a.) (pI)BIR01I FNYETETTSVIPAARK 1825.92 16  6.14 BIR02I PAARMFKAFILD 1378.74 12 9.18 BIR02J PAARMFKAFILEGDKLVPK 2130.26 19  9.72 BIR04PGTIKKISFPEGFPFKYV 2054.12 18  9.56 BIR07C SNEIKIVATPEGGSILK 1754.98 17 5.86 BIR09 ETLLRAVESYLLAHSDAY 2050.04 18  4.65 BIR09BKEMGETLLRAVESYLLAHS 2146.11 19  5.50 BIR12B AKYMVIQGEPGRVIRGK 1901.07 1710.28 BIR16A AERERIFKRFDANGEGK 2022.04 17  8.63

B) Solubility Testing

A series of matrices containing 260 mM trehalose and spanning a pH rangeof 3.0 to 7.0 plus a solution modified with 2 mM HCl were prepared asindicated in Appendix 2. The solubility of each of the nine peptides wasevaluated in each of the matrices in accordance with Appendix 1. Wheresolubility was achieved initially, but the peptide precipitated out ofsolution subsequently then an additional quantity of the relevant matrixwas added to try and achieve solubility of the peptide at ca. 200 μM.

Details of the Birch reference peptides used are indicated in Table 9-2.All peptides were manufactured by Bachem AG, Bubendorf, Switzerland.

TABLE 9-2 Details of Birch peptides Batch MW Peptide Peptide Peptide no.(Da) purity (%) content (%) BIR1I 1028882 1827.03 99.2 87.1 BIR02I1028883 1379.69 99.1 86.2 BIR02J 1028884 2131.61 97.4 84.1 BIR04 10288852055.45 98.1 88.3 BIR07C 1029310 1756.03 97.9 89.8 BIR09 1028886 2051.2897.5 86.9 BIR09B 1029311 2147.48 97.3 89.4 BIR12B 1028887 1902.30 97.385.9 BIR16A 1028888 2023.24 98.4 88.1

C) Results

The results of the solubility screening are displayed in tables 9-3 to9-8 below:

TABLE 3 2 mM HCl and 260 mM trehalose dihydrate, pH 2.65 Weight ofVolume Solubility Solubility 2 mM hydrochloric Acid peptide required [mgper Solubility [μmol Peptide Comments Solubility after 24 Hours ‘as is’(mg) (μL) ml ‘as is’] [mg per ml] per ml] BIR1I Completely dissolvedCompletely dissolved 0.975 50 19.50 16.849 9.222 BIR02I Completelydissolved Completely dissolved 1.175 50 23.50 20.075 14.550 BIR02JCompletely dissolved Completely dissolved 1.180 50 23.60 19.332 9.069BIR04 Completely dissolved Completely dissolved 1.025 50 20.50 17.7588.639 BIR07C Completely dissolved Completely dissolved 1.148 100 11.4810.093 5.747 BIR09 Completely dissolved • Completely dissolved • 1.1471250 0.92 0.777 0.379 filter debris filter debris BIR09B Completelydissolved Completely dissolved 1.220 100 12.20 10.612 4.942 BIR12BCompletely dissolved Completely dissolved 1.022 100 10.22 8.542 4.490BIR16A Completely dissolved Completely dissolved 1.243 50 24.86 21.55110.652

TABLE 4^(a) 10 mM sodium citrate and 260 mM trehalose dihydrate, pH 3.01Weight of Volume Solubility Solubility Citrate buffer pH 3.0 peptiderequired [mg per Solubility [μmol Peptide Comments Solubility after 24Hours ‘as is’ (mg) (μL) ml ‘as is’] [mg per ml] per ml] BIR1I Completelydissolved Completely dissolved 1.071 650 1.65 1.424 0.779 BIR02ICompletely dissolved Completely dissolved 1.112 1100 1.01 0.864 0.626BIR02J Completely dissolved in Precipitated out, 1.150 3000 0.38 0.3140.147 100 uL, redissolved in redissolved 3 mL after 24 hrs BIR04Completely dissolved Completely dissolved 1.129 50 22.58 19.559 9.516BIR07C Completely dissolved Completely dissolved 1.108 850 1.30 1.1460.653 BIR09 Undissolved in 1.5 mL Undissolved 1.111 3000 0.37 0.3140.153 diluted to 3 mL still undissolved BIR09B Completely dissolvedCompletely dissolved 1.162 100 11.62 10.108 4.707 BIR12B Completelydissolved Completely dissolved 1.094 200 5.47 4.572 2.403 BIR16ACompletely dissolved Completely dissolved 1.076 150 7.17 6.219 3.074

TABLE 4^(b) 10 mM sodium citrate and 260 mM trehalose dihydrate, pH 3.99Weight of Volume Solubility Solubility Citrate buffer pH 4.0 peptiderequired [mg per Solubility [μmol Peptide Comments Solubility after 24Hours ‘as is’ (mg) (μL) ml ‘as is’] [mg per ml] per ml] BIR1I Completelydissolved Completely dissolved 1.026 450 2.28 1.970 1.078 BIR02I Cloudysolution with Undissolved 1.051 3000 0.35 0.299 0.217 suspendedmaterial. Frothing on vortex BIR02J Completely dissolved in Precipitatedout, 1.155 3000 0.39 0.315 0.148 50 uL, redissolved in redissolved 3 mLafter 24 hrs BIR04 Completely dissolved Completely dissolved 1.018 5020.36 17.636 8.580 BIR07C Cloudy solution with Cloudy solution with1.126 3000 0.38 0.330 0.188 suspended material. suspended materialFrothing on vortex BIR09 Cloudy solution with Cloudy solution with 1.0743000 0.36 0.303 0.148 undissolved material undissolved material BIR09BCompletely dissolved Completely dissolved 1.140 100 11.40 9.916 4.618BIR12B Completely dissolved Completely dissolved 1.032 100 10.32 8.6264.534 BIR16A Completely dissolved Completely dissolved 1.123 150 7.496.490 3.208

TABLE 5 10 mM sodium citrate and 260 mM trehalose dihydrate, pH 5.02Weight of Volume Solubility Solubility Citrate buffer pH 5.0 peptiderequired [mg per Solubility [μmol Peptide Comments Solubility after 24Hours ‘as is’ (mg) (μL) ml ‘as is’] [mg per ml] per ml] BIR1I Completelydissolved Completely dissolved 1.036 450 2.30 1.989 1.089 BIR02I Cloudysolution with Completely dissolved 1.085 3000 0.36 0.309 0.224 suspendedmaterial BIR02J Completely dissolved in Precipitated out, 1.029 30000.34 0.281 0.132 150 uL, redissolved in redissolved 3 mL after 24 hrsBIR04 Completely dissolved Completely dissolved 1.076 50 21.52 18.6419.069 BIR07C Completely dissolved Completely dissolved 1.167 1350 0.860.760 0.433 BIR09 Clear solution with Clear solution with 1.115 30000.37 0.315 0.154 suspended material suspended material BIR09B Completelydissolved Completely dissolved 1.275 100 12.75 11.091 5.165 BIR12BCompletely dissolved Completely dissolved 1.167 200 5.84 4.877 2.564BIR16A Completely dissolved Completely dissolved 1.158 50 23.16 20.0779.923

TABLE 6 10 mM sodium citrate and 260 mM trehalose dihydrate, pH 6.01Weight of Volume Solubility Solubility Citrate buffer pH 6.0 peptiderequired [mg per Solubility [μmol Peptide Comments Solubility after 24Hours ‘as is’ (mg) (μL) ml ‘as is’] [mg per ml] per ml] BIR1I Completelydissolved Completely dissolved 1.117 150 7.45 6.434 3.522 BIR02I Cloudysolution with Cloudy solution with 1.143 3000 0.38 0.325 0.236 suspendedmaterial suspended material BIR02J Completely dissolved in Precipitatedout, 1.172 3000 0.39 0.320 0.150 100 uL, redissolved in redissolved 3 mLafter 24 hrs BIR04 Completely dissolved. Completely dissolved. 1.011 10010.11 8.758 4.261 Evidence of filter debris Filter debris BIR07CCompletely dissolved in Precipitated out, 1.045 3000 0.35 0.306 0.174350 uL, redissolved in redissolved 3 mL after 24 hrs BIR09 Completelydissolved completely dissolved 1.084 1050 1.03 0.875 0.426 BIR09RCompletely dissolved Completely dissolved 1.230 250 4.92 4.280 1.993BIR12B Completely dissolved. Completely dissolved. 1.026 450 2.28 1.9061.002 Evidence of filter debris Filter debris BIR16A Completelydissolved Completely dissolved 1.293 50 25.86 22.418 11.080

TABLE 7 10 mM potassium dihydrogen phosphate and 260 mM trehalosedihydrate, pH 6.03 Weight of Volume Solubility Solubility Phosphatebuffer pH 6.0 peptide required [mg per Solubility [μmol Peptide CommuteSolubility after 24 Hours ‘as is’ (mg) (μL) ml ‘as is’] [mg per ml] perml] BIR1I Completely dissolved Completely dissolved 1.107 50 22.1419.130 10.470 BIR02I Completely dissolved Completely dissolved 1.145 10011.45 9.781 7.089 BIR02J Completely dissolved Completely dissolved 1.112100 11.12 9.109 4.273 BIR04 Completely dissolved Completely dissolved0.986 100 9.86 8.541 4.155 BIR07C Completely dissolved in Precipitatedout, 1.245 3000 0.42 0.365 0.208 250 uL, redissolved in redissolved 3 mLafter 24 hrs BIR09 Cloudy solution with Cloudy solution with 1.037 30000.35 0.293 0.143 suspended material suspended material BIR09B Completelydissolved in Precipitated out, 1.086 3000 0.36 0.315 0.147 100 uL,redissolved in redissolved 3 mL after 24 hrs BIR12B Completely dissolvedCompletely dissolved 1.192 50 23.84 19.926 10.474 BIR16A Completelydissolved Completely dissolved 1.077 50 21.54 18.673 9.229

TABLE 8 10 mM potassium dihydrogen phosphate and 260 mM trehalosedihydrate, pH 7.03 Weight of Volume Solubility Solubility Phosphatebuffer pH 7.0 peptide required [mg per Solubility [μmol Peptide CommentsSolubility after 24 Hours ‘as is’ (mg) (μL) ml ‘as is’] [mg per ml] perml] BIR1I Completely dissolved Completely dissolved 1.039 50 20.7817.955 9.827 BIR02I Completely dissolved Completely dissolved 1.190 10011.90 10.165 7.368 BIR02J Completely dissolved Completely dissolved1.231 100 12.31 10.084 4.730 BIR04 Completely dissolved Completelydissolved 1.075 50 21.50 18.624 9.061 BIR07C Completely dissolvedCompletely dissolved 1.005 150 6.70 5.890 3.354 BIR09 Completelydissolved Completely dissolved 1.138 750 1.52 1.286 0.627 BIR09BCompletely dissolved Completely dissolved 1.085 50 21.70 18.876 8.790BIR12B Completely dissolved Completely dissolved 1.031 50 20.62 17.2349.060 BIR16A Completely dissolved Completely dissolved 1.099 50 21.9819.055 9.418

Example 9 Annex 1 Peptide Solubility Studies Solubility Methodology

The formulation vehicles were prepared and measurement of pH taken.

Weighing of peptides.

-   -   Approximately 1 mg was required for each evaluation.    -   Materials were dispensed into containers suitable for subsequent        solubility evaluation, i.e. clear glass HPLC vials (with screw        cap).

Evaluation of solubility (for each matrix).

-   -   Aliquots of matrix (50 to 100˜L) were added as required.    -   The peptide solubility was interpreted by visual inspection.    -   The description of the sample characteristics following addition        of each aliquot of the solvent was recorded.    -   Repeat visual assessment of solubility after 24 hours.

Where a peptide precipitated out of solution after 24 hours, additionalbuffer was added to produce a final concentration of ca. 0.2 mM (200nmol per mL should equate to roughly 0.35 mg/mL).

Calculation of peptide solubilities (initial evaluation).

-   -   Based on absolute amount of powder weighed.    -   Determination of molar concentration at which solubility was        achieved using peptide molecular masses and peptide content and        purity values.

Calculations

${{Solubility}\mspace{14mu} {mg}\text{/}{ml}\mspace{14mu} {‘{{as}\mspace{14mu} {is}}’}} = {\frac{{weight}({mg})}{{dilution}\left( {\mu l} \right)} \times 1000}$${{Solubility}\mspace{14mu} {mg}\text{/}{ml}} = {\frac{{weight}({mg})}{{dilution}\left( {\mu l} \right)} \times 1000 \times \% \mspace{14mu} {Content} \times \% \mspace{14mu} {Purity}}$${{Solubility}\mspace{14mu} {\mu mol}\text{/}{ml}} = {\frac{{weight}({mg})}{{dilution}\left( {\mu l} \right)} \times 1000 \times \% \mspace{14mu} {Content} \times \% \mspace{14mu} {Purity} \times \frac{1}{{Mol}\; {Wt}} \times 1000}$

Example 9 Annex 2 Buffers for Initial Solubility and Stability Screening

Each matrix was prepared at a concentration of 10 mM of the bufferingagent. Each buffer contained 260 mM Trehalose dihydrate (FW 378.3).

Preparation of Matrix

The procedure indicated is for the preparation of 100 mL of each buffer,but alternative volumes can be prepared by adjusting the quantities.

0.1M stock solutions of sodium citrate and potassium dihydrogenphosphate were prepared.

Weight of trehalose dihydrate equivalent to 260 mM was transferred to anappropriate mixing vessel containing 70-80 mL of distilled deionisedwater and allowed to dissolve.

10 mL of the appropriate stock 0.1M buffer solution was added to themixing vessel and stirred.

The pH of the matrix was adjusted to the desired value by adding 2 mMhydrochloric acid or 0.1M sodium hydroxide as required.

The solutions were finally diluted to 100 g weight and the pHre-assessed.

Buffers for initial solubility and stability screening, shown as Buffersalt or pH modifier/pH:

2 mM HCl and 260 mM trehalose dihydrate/pH 2.6510 mM sodium citrate and 260 mM trehalose dihydrate/pH 3.0110 mM sodium citrate and 260 mM trehalose dihydrate/pH 3.9910 mM sodium citrate and 260 mM trehalose dihydrate/pH 5.0210 mM sodium citrate and 260 mM trehalose dihydrate/pH 6.0110 mM potassium dihydrogen phosphate and 260 mM trehalose dihydrate/pH6.0310 mM potassium dihydrogen phosphate and 260 mM trehalose dihydrate/pH7.03

Example 10 Histamine Release Assay

The purpose of this assay was to identify compositions that are capableof activating blood basophils (as a surrogate for tissue mast cells)resulting in histamine release that may result in allergic reactionsduring therapy. A composition comprising a mixture of peptides thatinduce histamine release frequently may be considered unsuitable for useas a vaccine.

Histamine release requires the crosslinking of adjacent specific IgEmolecules on the surface of the basophil. The peptides being evaluatedwere small (11 to 18 amino acids in length) and should not, therefore,possess significant tertiary structure that would enable them to retainthe conformation of an IgE-binding epitope of the whole molecule.Furthermore, peptide monomers in solution, even if they are bound byIgE, should not be able to crosslink adjacent IgE molecules.

Histamine release from fresh peripheral whole blood from birch allergicsubjects was evaluated. Peripheral blood basophils were used as asurrogate for tissue mast cells which were not practical to assay. Bloodwas incubated in vitro with mixtures of peptides identified as suitablebased on the results of Examples 1 to 9 above. Specifically, thefollowing mixtures were assayed:

Mix 1—BIR01I, BIR02J, BIR04, BIR12B, BIR16A, BIR07C Mix 2—BIR01I,BIR02J, BIR04, BIR12B, BIR16A, BIR07C, BIR09 Mix 3—BIR01I, BIR02J,BIR04, BIR12B, BIR16A, BIR07C, BIR09B Mix 4—BIR01I, BIR02I, BIR04,BIR12B, BIR16A, BIR07C Mix 5—BIR01I, BIR02I, BIR04, BIR12B, BIR16A,BIR07C, BIR09 Mix 6—BIR01I, BIR02I, BIR04, BIR12B, BIR16A, BIR07C,BIR09B

Histamine release in response to whole birch allergen extract wasmeasured in each subject to confirm basophil sensitisation. A positivecontrol, representing total histamine release, generated byfreeze/thawing the cells twice, was included in each assay. A negativecontrol for spontaneous histamine release was generated by incubatingcells in buffer only.

The assay was performed using the Immunotech Histamine ReleaseImmunoassay kit according to the manufacturer's instructions. Followingthe in vitro challenge of blood basophils with peptide mixtures, wholeallergen or buffer in microtitre plate wells, supernatants were removedand the histamine in the samples converted to acyl histamine. Acylatedsamples were tested by a competitive acyl histamine ELISA.

Peptide mixtures were assayed for their ability to induce histaminerelease over a 5 log 10 range (1 to 10,000 ng/ml). The concentrationrange assayed was selected based on theoretical in vivo doses of peptidethat may be achieved during therapy. For example, a 31 μg dose(approximately 3 nmol/peptide equivalent) of each peptide entering ablood volume of 5 litres, would result in a blood concentration of 6ng/ml, at the lower end of the histamine release assay dose range. Thewhole birch allergen extract was used over the same concentration range.

Single measurements were performed for each dilution. After completionof the ELISA, individual histamine levels were determined byinterpolation from the standard curve generated in the ELISA assay.Results from samples were adjusted to allow for dilution. Where two ormore consecutive dilutions of a peptide/allergen preparationelicited >15% of the total histamine release seen in the freeze thawedpositive control (>15% of positive control), or where a single valueof >15% of positive control was achieved at the highest concentrationtested (10 μg/mL for peptides), this was considered a “positivehistamine release”.

A total of 40 histamine release assays were completed during the study.Of these 5 assays were rejected because of failure to meet appropriateQC controls, e.g. due to unacceptably high levels (>15% of positivecontrol) of spontaneous release in the medium plus buffer negativecontrol wells.

The mixtures tested all showed good histamine release properties. Thestudy findings are summarised as follows: (WA=whole allergen)

Mix Peptide conc: μg/ml Average % histamine release 1 10 1% 1 1 2% 1 0.13% 1 0.01 3% 1 0.001 2% 2 10 2% 2 1 2% 2 0.1 3% 2 0.01 3% 2 0.001 2% 310 4% 3 1 2% 3 0.1 3% 3 0.01 2% 3 0.001 2% 4 10 2% 4 1 2% 4 0.1 2% 40.01 1% 4 0.001 1% 5 10 2% 5 1 2% 5 0.1 2% 5 0.01 2% 5 0.001 2% 6 10 3%6 1 2% 6 0.1 1% 6 0.01 1% 6 0.001 2% WA 10 65% WA 1 38% WA 0.1 38% WA0.01 42% WA 0.001 43%

1. A composition of matter suitable for use in preventing or treatingallergy to birch pollen, said composition being selected from the groupconsisting of: a) a composition comprising: i) at least one of thepolypeptides of SEQ ID NO: 74 (BIR12B; AKYMVIQGEPGRVIRGK), SEQ ID NO: 72(BIR11; FPQFKPQEITGIMK), SEQ ID NO: 71 (BIR10; GSVWAQSSSFPQFK), SEQ IDNO: 73 (BIR12A; PTGMFVAGAKYMVIQGR), SEQ ID NO: 75 (BIR13;IKYMVIQGEAGAVIRGK) and SEQ ID NO: 76 (BIR14; EAGAVIRGKKGSGGIT), or avariant of any thereof, and ii) at least one of the polypeptides of SEQID NO: 53 (Bir02J; PAARMFKAFILEGDKLVPK), SEQ ID NO: 48 (Bir01I;FNYETETTSVIPAARK), SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV), SEQ ID NO:67 (Bir09; ETLLRAVESYLLAHSDAY), SEQ ID NO: 60 (BIR07;SNEIKIVATPDGGSILK), and SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK), or avariant of any thereof, wherein a variant of a polypeptide is: I) up to30 amino acids in length and comprises the sequence of the saidpolypeptide, or II) 9 to 30 amino acids in length and comprises asequence that has at least 65% homology to the sequence of saidpolypeptide, which sequence is capable of tolerising to saidpolypeptide; or III) 9 to 30 amino acids in length and comprises asequence of at least 9 contiguous amino acids of the sequence of saidpolypeptide, or a sequence that has at least 65% homology to said atleast 9 contiguous amino acids, which sequence of at least 9 contiguousamino acids or homologous sequence is capable of tolerising to saidpolypeptide; b) a composition comprising at least one polynucleotidesequence which when expressed causes the production of a composition asdefined in (i); c) a vector comprising four or more polynucleotidesequences which each encode a different polypeptide as defined in (i);d) a product comprising: i) at least one of the polypeptides of SEQ IDNO: 74 (BIR12B; AKYMVIQGEPGRVIRGK), SEQ ID NO: 72 (BIR11;FPQFKPQEITGIMK), SEQ ID NO: 71 (BIR10; GSVWAQSSSFPQFK), SEQ ID NO: 73(BIR12A; PTGMFVAGAKYMVIQGR), SEQ ID NO: 75 (BIR13; IKYMVIQGEAGAVIRGK)and SEQ ID NO: 76 (BIR14; EAGAVIRGKKGSGGIT), or a variant of anythereof, and ii) at least one of the polypeptides of SEQ ID NO: 53(Bir02J; PAARMFKAFILEGDKLVPK), SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK),SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV), SEQ ID NO: 67 (Bir09;ETLLRAVESYLLAHSDAY), SEQ ID NO: 60 (BIR07; SNEIKIVATPDGGSILK), and SEQID NO: 63 (Bir07C; SNEIKIVATPEGGSILK), or a variant of any thereof,wherein a variant of a polypeptide is as defined in claim 1(a) (I), (II)or (III), and wherein each different polypeptide is for simultaneous,separate or sequential use in preventing or treating allergy to birchpollen; and e) a pharmaceutical formulation comprising a compositionaccording to (a) or (b); a vector according to (c); or a productaccording to (d); and a pharmaceutically acceptable carrier or diluent,and optionally one or more adjuvants selected from a glucocorticoid,vitamin D and rapamycin.
 2. A composition according to claim 1(a), whichis selected from the group consisting of: i) a composition of claim 1(a)which further comprises at least one additional polypeptide of claim1(a)(i) or (ii) or variant thereof not selected in claim 1(a); ii) acomposition of claim 1(a) which further comprises at least oneadditional polypeptide selected from the group consisting of SEQ ID NO:77 (BIR15; SLNTLRLRRIFDLFDK) and SEQ ID NO:78 (BIR16A;AERERIFKRFDANGEGK), or a variant of any thereof; and iii) a compositionof claim 1(a) which comprises: (a) the polypeptide SEQ ID NO: 74(Bir12B; AKYMVIQGEPGRVIRGK), or a variant thereof; (b) the polypeptideSEQ ID NO: 53 (Bir02J; PAARMFKAFILEGDKLVPK), or a variant thereof; and(c) the polypeptide SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK) or avariant thereof; wherein a variant of a polypeptide is as defined inclaim 1(a) (I), (II) or (III), preferably wherein; when said polypeptideis SEQ ID NO: 48 said variant of SEQ ID NO: 48 is SEQ ID NO: 45 (Bir01F;FNYETEATSVIPAARK), SEQ ID NO: 46 (Bir01G; FNYEIEATSVIPAARK) or SEQ IDNO: 47 (Bir01H; FNYEIETTSVIPAARK); and when said polypeptide is SEQ IDNO: 53, said variant of SEQ ID NO: 53 is SEQ ID NO: 50 (Bir02E;PAARLFKAFILEGDTLIPK), SEQ ID NO: 51 (Bir02G; PAARLFKAFILEGDNLIPK), SEQID NO: 52 (Bir02I; PAARMFKAFILD) or SEQ ID NO: 49 (Bir02D;PAARMFKAFILDGDKLVPK); and when said polypeptide is SEQ ID NO: 67, saidvariant of SEQ ID NO: 67 is selected from SEQ ID NO: 68 (Bir09A;GETLLRAVESYLLAHS), SEQ ID NO: 69 (Bir09B; KEMGETLLRAVESYLLAHS) or SEQ IDNO: 70 (Bir09C; KEKGETLLRAVESYLLAHS); and when said polypeptide is SEQID NO: 78 said variant of SEQ ID NO: 78 is SEQ ID NO: 79 (Bir16B;AERERIFKRFDAGGEGK). 3-5. (canceled)
 6. A composition suitable for use inpreventing or treating allergy to birch pollen comprising at least threedifferent polypeptides, selected from: (a) SEQ ID NO: 74 (Bir12B;AKYMVIQGEPGRVIRGK), or a variant thereof; (b) SEQ ID NO: 53 (Bir02J;PAARMFKAFILEGDKLVPK), or a variant thereof; (c) SEQ ID NO: 48 (Bir01I;FNYETETTSVIPAARK) or a variant thereof; (d) SEQ ID NO: 54 (Bir04;PGTIKKISFPEGFPFKYV) or a variant thereof; (e) SEQ ID NO: 67 (Bir09;ETLLRAVESYLLAHSDAY) or a variant thereof; (f) SEQ ID NO: 78 (Bir16A;AERERIFKRFDANGEGK) or a variant thereof; (g) SEQ ID NO: 60 (Bir07;SNEIKIVATPDGGSILK) or a variant thereof; (h) SEQ ID NO: 63 (Bir07C;SNEIKIVATPEGGSILK) or a variant thereof; (i) SEQ ID NO: 72 (Bir01I;FPQFKPQEITGIMK) or a variant thereof; (j) SEQ ID NO: 77 (Bir15;SLNTLRLRRIFDLFDK) or a variant thereof; wherein a variant of apolypeptide is: I) up to 30 amino acids in length and comprises thesequence of said polypeptide, or II) 9 to 30 amino acids in length andcomprises a sequence that has at least 65% homology to the sequence ofsaid polypeptide, which sequence is capable of tolerising to saidpolypeptide; or III) 9 to 30 amino acids in length and comprises asequence of at least 9 contiguous amino acids of the sequence of saidpolypeptide, or a sequence that has at least 65% homology to said atleast 9 contiguous amino acids, which sequence of at least 9 contiguousamino acids or homologous sequence is capable of tolerising to saidpolypeptide.
 7. A composition according to claim 6, which is selectedfrom the group consisting of: i) a composition comprising: a) thepolypeptide SEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or a variantthereof; b) at least one of the polypeptides SEQ ID NO: 53 (Bir02J;PAARMFKAFILEGDKLVPK) and SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK), or avariant of any thereof; and c) at least one additional polypeptide of a)to j); ii) a composition comprising the polypeptide SEQ ID NO: 74(Bir12B; AKYMVIQGEPGRVIRGK) or a variant thereof, the polypeptide SEQ IDNO: 53 (Bir02J; PAARMFKAFILEGDKLVPK) or a variant thereof, thepolypeptide SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK) or a variantthereof, and at least one additional polypeptide of a) to j); iii) acomposition comprising the polypeptide SEQ ID NO: 74 (Bir12B;AKYMVIQGEPGRVIRGK) or a variant thereof, the polypeptide SEQ ID NO: 53(Bir02J; PAARMFKAFILEGDKLVPK) or a variant thereof, the polypeptide SEQID NO: 48 (Bir01I; FNYETETTSVIPAARK) or a variant thereof, thepolypeptide SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a variantthereof, the polypeptide SEQ ID NO: 67 (Bir09; ETLLRAVESYLLAHSDAY) or avariant thereof, the polypeptide SEQ ID NO: 63 (Bir07C;SNEIKIVATPEGGSILK) or a variant thereof, and the polypeptide SEQ ID NO:78 (Bir16A; AERERIFKRFDANGEGK) or a variant thereof, and optionally nofurther polypeptides derived from a birch pollen allergen; iv) acomposition comprising the polypeptide SEQ ID NO: 74 (Bir12B;AKYMVIQGEPGRVIRGK) or a variant thereof, the polypeptide SEQ ID NO: 53(Bir02J; PAARMFKAFILEGDKLVPK) or a variant thereof, the polypeptide SEQID NO: 48 (Bir01I; FNYETETTSVIPAARK) or a variant thereof, thepolypeptide SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a variantthereof, the polypeptide SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK) or avariant thereof, the polypeptide SEQ ID NO: 78 (Bir16A;AERERIFKRFDANGEGK) or a variant thereof, and the polypeptide SEQ ID NO:69 (Bir09B; KEMGETLLRAVESYLLAHS), and optionally no further polypeptidesderived from a birch pollen allergen; and v) a composition comprisingthe polypeptide SEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or a variantthereof, the polypeptide SEQ ID NO: 53 (Bir02J; PAARMFKAFILEGDKLVPK) ora variant thereof, the polypeptide SEQ ID NO: 48 (Bir01I;FNYETETTSVIPAARK) or a variant thereof, the polypeptide SEQ ID NO: 54(Bir04; PGTIKKISFPEGFPFKYV) or a variant thereof, the polypeptide SEQ IDNO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a variant thereof, and thepolypeptide SEQ ID NO: 78 (Bir16A; AERERIFKRFDANGEGK) or a variantthereof, and optionally no further polypeptides derived from a birchpollen allergen; wherein a variant of a polypeptide is as defined inclaim 3 (I), (II) or (III), preferably wherein: when said polypeptide isSEQ ID NO: 48 said variant of SEQ ID NO: 48 is SEQ ID NO: 45 (Bir01F;FNYETEATSVIPAARK), SEQ ID NO: 46 (Bir01G; FNYEIEATSVIPAARK) or SEQ IDNO: 47 (Bir01H; FNYEIETTSVIPAARK); and when said polypeptide is SEQ IDNO: 53, said variant of SEQ ID NO: 53 is SEQ ID NO: 50 (Bir02E;PAARLFKAFILEGDTLIPK), SEQ ID NO: 51 (Bir02G; PAARLFKAFILEGDNLIPK), SEQID NO: 52 (Bir02I; PAARMFKAFILD) or SEQ ID NO: 49 (Bir02D;PAARMFKAFILDGDKLVPK); and when said polypeptide is SEQ ID NO: 67, saidvariant of SEQ ID NO: 67 is selected from SEQ ID NO: 68 (Bir09A;GETLLRAVESYLLAHS), SEQ ID NO: 69 (Bir09B; KEMGETLLRAVESYLLAHS) or SEQ IDNO: 70 (Bir09C; KEKGETLLRAVESYLLAHS); and when said polypeptide is SEQID NO: 78, said variant of SEQ ID NO: 78 is SEQ ID NO: 79 (Bir 16B;AERERIFKRFDAGGEGK). 8-12. (canceled)
 13. The composition according toclaim 1(a), wherein the composition: is capable of tolerising at least50% or at least 60% of a panel of birch pollen allergic individuals inthe population and/or comprises at least one further polypeptide up to atotal of thirteen unique/different polypeptides, wherein the furtherpolypeptides: (a) comprise a sequence having at least 65% sequenceidentity to at least 9 or more contiguous amino acids in any of SEQ IDNOs: 1 to 80 not selected above; and (b) are 9 to 30 amino acids inlength.
 14. The composition according to claim 1(a), comprising at leastone said polypeptide which is 9 to 20 or 13 to 17 amino acids in lengthand/or wherein said polypeptide has at least 70% sequence identity toany of SEQ ID NOS: 1 to
 80. 15. The composition according to claim 1(a),wherein one or more of the polypeptides have one or more modificationsselected from the following: (i) N terminal acetylation; (ii) C terminalamidation; (iii) one or more hydrogens on the side chain amines ofArginine and/or Lysine replaced with a methylene group; (iv)glycosylation; and (v) phosphorylation.
 16. The composition according toclaim 1(a) wherein at least one of the polypeptides has been engineeredto be soluble such that it comprises: i) N terminal to the residues ofthe polypeptide which flank a T cell epitope: one to six contiguousamino acids corresponding to the two to six contiguous amino acidsimmediately N terminal to said residues in the sequence of the proteinfrom which the polypeptide derives; and/or ii) C terminal to theresidues of the polypeptide which flank a T cell epitope: one to sixcontiguous amino acids corresponding to the one to six contiguous aminoacids immediately C terminal to the said residues in the sequence of theprotein from which the polypeptide derives; or iii) both N and Cterminal to the residues of the polypeptide which flank a T cellepitope, at least one amino acid selected from arginine, lysine,histidine, glutamate and aspartate, wherein the polypeptide has asolubility of at least 3.5 mg/ml and the T cell epitope has a solubilityof less than 3.5 mg/ml.
 17. The composition according to claim 1(a)wherein at least one of the polypeptides has been engineered to besoluble such that additionally: i) any cysteine residues in the nativesequence of the polypeptide are replaced with serine or 2-aminobutyricacid; and/or ii) any hydrophobic residues in the up to three amino acidsat the N or C terminus of the native sequence of the polypeptide, whichare not comprised in a T cell epitope, are deleted; and/or iii) any twoconsecutive amino acids comprising the sequence Asp-Gly in the up tofour amino acids at the N or C terminus of the native sequence of thepolypeptide, which are not comprised in a T cell epitope, are deleted;and/or iv) one or more positively charged residues are added at the Nand/or C terminus of the native sequence of the polypeptide.
 18. Thecomposition according to claim 1(a) wherein each polypeptide has aconcentration in the range of 0.03 to 200 nmol/ml, 0.3 to 200 nmol/ml,50 to 200 nmol/ml or 30 to 120 nmol/ml. 19-23. (canceled)
 24. Thecomposition according to claim 1(e), wherein said composition of claim1(a) or (b), said vector of claim 1(c) or said product of claim 1(d) isformulated for oral administration, nasal administration, topicaladministration, subcutaneous administration, sublingual administration,intradermal administration, buccal administration, epidermaladministration, or for administration by inhalation, by injection, or bya patch.
 25. The composition according to claim 1 wherein saidcomposition of claim 1(a) or product of claim 1(d), additionallycomprises a further polypeptide allergen for use in tolerising anindividual to the further polypeptide allergen.
 26. An in vitro methodof determining whether T cells recognize a composition according toclaim 1(a), comprising contacting said T cells with said composition anddetecting whether said T cells are stimulated by said composition.
 27. Amethod according to claim 26 which is carried out to determine whetheran individual has, or is at risk of having, an allergy to birch pollen.28. A method of preventing or treating allergy to birch pollen, themethod comprising administering to a subject in need of such treatment atherapeutically effective amount of: (A) a composition comprising: i) atleast one of the polypeptides of SEQ ID NO: 74 (BIR12B;AKYMVIQGEPGRVIRGK), SEQ ID NO: 72 (BIR11; FPQFKPQEITGIMK), SEQ ID NO: 71(BIR10; GSVWAQSSSFPQFK), SEQ ID NO: 73 (BIR12A; PTGMFVAGAKYMVIQGR), SEQID NO: 75 (BIR13; IKYMVIQGEAGAVIRGK) and SEQ ID NO: 76 (BIR14;EAGAVIRGKKGSGGIT), or a variant of any thereof, and ii) at least one ofthe polypeptides of SEQ ID NO: 53 (Bir02J; PAARMFKAFILEGDKLVPK), SEQ IDNO: 48 (Bir01I; FNYETETTSVIPAARK), SEQ ID NO: 54 (Bir04;PGTIKKISFPEGFPFKYV), SEQ ID NO: 67 (Bir09; ETLLRAVESYLLAHSDAY), SEQ IDNO: 60 (BIR07; SNEIKIVATPDGGSILK), and SEQ ID NO: 63 (Bir07C;SNEIKIVATPEGGSILK), or a variant of any thereof, wherein a variant of apolypeptide is: I) up to 30 amino acids in length and comprises thesequence of the said polypeptide, or II) 9 to 30 amino acids in lengthand comprises a sequence that has at least 65% homology to the sequenceof said polypeptide, which sequence is capable of tolerising to saidpolypeptide; or III) 9 to 30 amino acids in length and comprises asequence of at least 9 contiguous amino acids of the sequence of saidpolypeptide, or a sequence that has at least 65% homology to said atleast 9 contiguous amino acids, which sequence of at least 9 contiguousamino acids or homologous sequence is capable of tolerising to saidpolypeptide. or (B) a composition comprising at least three differentpolypeptides, selected from: (a) SEQ ID NO: 74 (Bir12B;AKYMVIQGEPGRVIRGK), or a variant thereof; (b) SEQ ID NO: 53 (Bir02J;PAARMFKAFILEGDKLVPK), or a variant thereof; (c) SEQ ID NO: 48 (Bir01I;FNYETETTSVIPAARK) or a variant thereof; (d) SEQ ID NO: 54 (Bir04;PGTIKKISFPEGFPFKYV) or a variant thereof; (e) SEQ ID NO: 67 (Bir09;ETLLRAVESYLLAHSDAY) or a variant thereof; (f) SEQ ID NO: 78 (Bir16A;AERERIFKRFDANGEGK) or a variant thereof; (g) SEQ ID NO: 60 (Bir07;SNEIKIVATPDGGSILK) or a variant thereof; (h) SEQ ID NO: 63 (Bir07C;SNEIKIVATPEGGSILK) or a variant thereof; (i) SEQ ID NO: 72 (Bir011;FPQFKPQEITGIMK) or a variant thereof; (j) SEQ ID NO: 77 (Bir15;SLNTLRLRRIFDLFDK) or a variant thereof; wherein a variant of apolypeptide is: I) up to 30 amino acids in length and comprises thesequence of said polypeptide, or II) 9 to 30 amino acids in length andcomprises a sequence that has at least 65% homology to the sequence ofsaid polypeptide, which sequence is capable of tolerising to saidpolypeptide; or III) 9 to 30 amino acids in length and comprises asequence of at least 9 contiguous amino acids of the sequence of saidpolypeptide, or a sequence that has at least 65% homology to said atleast 9 contiguous amino acids, which sequence of at least 9 contiguousamino acids or homologous sequence is capable of tolerising to saidpolypeptide.
 29. A composition according to claim 1, which is selectedfrom the group consisting of: i) a composition comprising thepolypeptide of SEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 53 (Bir02J;PAARMFKAFILEGDKLVPK) or a said variant thereof, the polypeptide of SEQID NO:48 (Bir01I; FNYETETTSVIPAARK) or a said variant thereof, thepolypeptide of SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a saidvariant thereof, the polypeptide of SEQ ID NO: 67 (Bir09;ETLLRAVESYLLAHSDAY) or a said variant thereof, the polypeptide of SEQ IDNO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a said variant thereof, and thepolypeptide of SEQ ID NO: 78 (Bir16A; AERERIFKRFDANGEGK) or a saidvariant thereof; and optionally no further polypeptides derived from abirch pollen allergen; ii) a composition comprising the polypeptide ofSEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or a said variant thereof, thepolypeptide of SEQ ID NO: 53 (Bir02J; PAARMFKAFILEGDKLVPK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 48 (Bir01I;FNYETETTSVIPAARK) or a said variant thereof, the polypeptide of SEQ IDNO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a said variant thereof, thepolypeptide of SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 78 (Bir16A;AERERIFKRFDANGEGK) or a said variant thereof, and the polypeptide of SEQID NO: 69 (Bir09B; KEMGETLLRAVESYLLAHS) or a said variant thereof, andoptionally no further polypeptides derived from a birch pollen allergen;iii) a composition comprising the polypeptide of SEQ ID NO: 74 (Bir12B;AKYMVIQGEPGRVIRGK) or a said variant thereof, the polypeptide of SEQ IDNO: 53 (Bir02J; PAARMFKAFILEGDKLVPK) or a said variant thereof, thepolypeptide of SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 54 (Bir04;PGTIKKISFPEGFPFKYV) or a said variant thereof, the polypeptide of SEQ IDNO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a said variant thereof, and thepolypeptide of SEQ ID NO: 78 (Bir16A; AERERIFKRFDANGEGK) or a saidvariant thereof, and optionally no further polypeptides derived from abirch pollen allergen; iv) a composition comprising the polypeptide ofSEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or a said variant thereof, thepolypeptide of SEQ ID NO: 52 (Bir02I; PAARMFKAFILD) or a said variantthereof, the polypeptide of SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK) ora said variant thereof, the polypeptide of SEQ ID NO: 54 (Bir04;PGTIKKISFPEGFPFKYV) or a said variant thereof, the polypeptide of SEQ IDNO: 67 (Bir09; ETLLRAVESYLLAHSDAY) or a said variant thereof, thepolypeptide of SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a saidvariant thereof, and the polypeptide of SEQ ID NO: 78 (Bir16A;AERERIFKRFDANGEGK) or a said variant thereof; v) a compositioncomprising the polypeptide of SEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK)or a said variant thereof, the polypeptide of SEQ ID NO: 52 (Bir02I;PAARMFKAFILD) or a said variant thereof, the polypeptide of SEQ ID NO:48 (Bir01I; FNYETETTSVIPAARK) or a said variant thereof, the polypeptideof SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a said variant thereof,the polypeptide of SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 78 (Bir16A;AERERIFKRFDANGEGK) or a said variant thereof, and the polypeptide of SEQID NO: 69 (Bir09B; KEMGETLLRAVESYLLAHS) or a said variant thereof; andvi) a composition comprising the polypeptide of SEQ ID NO: 74 (Bir12B;AKYMVIQGEPGRVIRGK) or a said variant thereof, the polypeptide of SEQ IDNO: 52 (Bir02I; PAARMFKAFILD) or a said variant thereof, the polypeptideof SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK) or a said variant thereof,the polypeptide of SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a saidvariant thereof, the polypeptide of SEQ ID NO: 63 (Bir07C;SNEIKIVATPEGGSILK) or a said variant thereof, and the polypeptide of SEQID NO: 78 (Bir16A; AERERIFKRFDANGEGK).
 30. The method according to claim28, wherein the composition is selected from the group consisting of: i)a composition comprising the polypeptide of SEQ ID NO: 74 (Bir12B;AKYMVIQGEPGRVIRGK) or a said variant thereof, the polypeptide of SEQ IDNO: 53 (Bir02J; PAARMFKAFILEGDKLVPK) or a said variant thereof, thepolypeptide of SEQ ID NO:48 (Bir01I; FNYETETTSVIPAARK) or a said variantthereof, the polypeptide of SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) ora said variant thereof, the polypeptide of SEQ ID NO: 67 (Bir09;ETLLRAVESYLLAHSDAY) or a said variant thereof, the polypeptide of SEQ IDNO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a said variant thereof, and thepolypeptide of SEQ ID NO: 78 (Bir16A; AERERIFKRFDANGEGK) or a saidvariant thereof; and optionally no further polypeptides derived from abirch pollen allergen; ii) a composition comprising the polypeptide ofSEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or a said variant thereof, thepolypeptide of SEQ ID NO: 53 (Bir02J; PAARMFKAFILEGDKLVPK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 48 (Bir01I;FNYETETTSVIPAARK) or a said variant thereof, the polypeptide of SEQ IDNO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a said variant thereof, thepolypeptide of SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 78 (Bir16A;AERERIFKRFDANGEGK) or a said variant thereof, and the polypeptide of SEQID NO: 69 (Bir09B; KEMGETLLRAVESYLLAHS) or a said variant thereof, andoptionally no further polypeptides derived from a birch pollen allergen;iii) a composition comprising the polypeptide of SEQ ID NO: 74 (Bir12B;AKYMVIQGEPGRVIRGK) or a said variant thereof, the polypeptide of SEQ IDNO: 53 (Bir02J; PAARMFKAFILEGDKLVPK) or a said variant thereof, thepolypeptide of SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 54 (Bir04;PGTIKKISFPEGFPFKYV) or a said variant thereof, the polypeptide of SEQ IDNO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a said variant thereof, and thepolypeptide of SEQ ID NO: 78 (Bir16A; AERERIFKRFDANGEGK) or a saidvariant thereof, and optionally no further polypeptides derived from abirch pollen allergen; iv) a composition comprising the polypeptide ofSEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or a said variant thereof, thepolypeptide of SEQ ID NO: 52 (Bir02I; PAARMFKAFILD) or a said variantthereof, the polypeptide of SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK) ora said variant thereof, the polypeptide of SEQ ID NO: 54 (Bir04;PGTIKKISFPEGFPFKYV) or a said variant thereof, the polypeptide of SEQ IDNO: 67 (Bir09; ETLLRAVESYLLAHSDAY) or a said variant thereof, thepolypeptide of SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a saidvariant thereof, and the polypeptide of SEQ ID NO: 78 (Bir16A;AERERIFKRFDANGEGK) or a said variant thereof; v) a compositioncomprising the polypeptide of SEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK)or a said variant thereof, the polypeptide of SEQ ID NO: 52 (Bir02I;PAARMFKAFILD) or a said variant thereof, the polypeptide of SEQ ID NO:48 (Bir01I; FNYETETTSVIPAARK) or a said variant thereof, the polypeptideof SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a said variant thereof,the polypeptide of SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 78 (Bir16A;AERERIFKRFDANGEGK) or said variant thereof, and the polypeptide of SEQID NO: 69 (Bir09B; KEMGETLLRAVESYLLAHS) or a said variant thereof; andvi) a composition comprising the polypeptide of SEQ ID NO: 74 (Bir12B;AKYMVIQGEPGRVIRGK) or a said variant thereof, the polypeptide of SEQ IDNO: 52 (Bir02I; PAARMFKAFILD) or a said variant thereof, the polypeptideof SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK) or a said variant thereof,the polypeptide of SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a saidvariant thereof, the polypeptide of SEQ ID NO: 63 (Bir07C;SNEIKIVATPEGGSILK) or a said variant thereof, and the polypeptide of SEQID NO: 78 (Bir16A; AERERIFKRFDANGEGK).
 31. A method of preparing apharmaceutical formulation, the method comprising combining with apharmaceutically acceptable carrier or diluent i) at least one of thepolypeptides of SEQ ID NO: 74 (BIR12B; AKYMVIQGEPGRVIRGK), SEQ ID NO: 72(BIR11; FPQFKPQEITGIMK), SEQ ID NO: 71 (BIR10; GSVWAQSSSFPQFK), SEQ IDNO: 73 (BIR12A; PTGMFVAGAKYMVIQGR), SEQ ID NO: 75 (BIR13;IKYMVIQGEAGAVIRGK and SEQ ID NO: 76 (BIR14; EAGAVIRGKKGSGGIT), or avariant of any thereof, and ii) at least one of the polypeptides of SEQID NO: 53 (Bir02J; PAARMFKAFILEGDKLVPK), SEQ ID NO: 48 (Bir01I;FNYETETTSVIPAARK), SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV), SEQ ID NO:67 (Bir09; ETLLRAVESYLLAHSDAY), SEQ ID NO: 60 (BIR07;SNEIKIVATPDGGSILK), and SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK), or avariant of any thereof, wherein a variant of a polypeptide is: I) up to30 amino acids in length and comprises the sequence of the saidpolypeptide, or II) 9 to 30 amino acids in length and comprises asequence that has at least 65% homology to the sequence of saidpolypeptide, which sequence is capable of tolerising to saidpolypeptide; or III) 9 to 30 amino acids in length and comprises asequence of at least 9 contiguous amino acids of the sequence of saidpolypeptide, or a sequence that has at least 65% homology to said atleast 9 contiguous amino acids, which sequence of at least 9 contiguousamino acids or homologous sequence is capable of tolerising to saidpolypeptide.
 32. A method according to claim 31, the method comprisingcombining with a pharmaceutically acceptable carrier or diluent i) thepolypeptide of SEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 53 (Bir02J;PAARMFKAFILEGDKLVPK) or a said variant thereof, the polypeptide of SEQID NO:48 (Bir01I; FNYETETTSVIPAARK) or a said variant thereof, thepolypeptide of SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a saidvariant thereof, the polypeptide of SEQ ID NO: 67 (Bir09;ETLLRAVESYLLAHSDAY) or a said variant thereof, the polypeptide of SEQ IDNO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a said variant thereof, and thepolypeptide of SEQ ID NO: 78 (Bir16A; AERERIFKRFDANGEGK) or a saidvariant thereof; and optionally no further polypeptides derived from abirch pollen allergen; ii) the polypeptide of SEQ ID NO: 74 (Bir12B;AKYMVIQGEPGRVIRGK) or a said variant thereof, the polypeptide of SEQ IDNO: 53 (Bir02J; PAARMFKAFILEGDKLVPK) or a said variant thereof, thepolypeptide of SEQ ID NO: 48 (Bir01I; FNYETETTSVIPAARK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 54 (Bir04;PGTIKKISFPEGFPFKYV) or a said variant thereof, the polypeptide of SEQ IDNO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a said variant thereof, thepolypeptide of SEQ ID NO: 78 (Bir16A; AERERIFKRFDANGEGK) or a saidvariant thereof, and the polypeptide of SEQ ID NO: 69 (Bir09B;KEMGETLLRAVESYLLAHS) or a said variant thereof, and optionally nofurther polypeptides derived from a birch pollen allergen; iii) thepolypeptide of SEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 53 (Bir02J;PAARMFKAFILEGDKLVPK) or a said variant thereof, the polypeptide of SEQID NO: 48 (Bir01I; FNYETETTSVIPAARK) or a said variant thereof, thepolypeptide of SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a saidvariant thereof, the polypeptide of SEQ ID NO: 63 (Bir07C;SNEIKIVATPEGGSILK) or a said variant thereof, and the polypeptide of SEQID NO: 78 (Bir16A; AERERIFKRFDANGEGK) or a said variant thereof, andoptionally no further polypeptides derived from a birch pollen allergen;iv) the polypeptide of SEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or asaid variant thereof, the polypeptide of SEQ ID NO: 52 (Bir02I;PAARMFKAFILD) or a said variant thereof, the polypeptide of SEQ ID NO:48 (Bir01I; FNYETETTSVIPAARK) or a said variant thereof, the polypeptideof SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a said variant thereof,the polypeptide of SEQ ID NO: 67 (Bir09; ETLLRAVESYLLAHSDAY) or a saidvariant thereof, the polypeptide of SEQ ID NO: 63 (Bir07C;SNEIKIVATPEGGSILK) or a said variant thereof, and the polypeptide of SEQID NO: 78 (Bir16A; AERERIFKRFDANGEGK) or a said variant thereof; v) thepolypeptide of SEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 52 (Bir02I; PAARMFKAFILD)or a said variant thereof, the polypeptide of SEQ ID NO: 48 (Bir01I;FNYETETTSVIPAARK) or a said variant thereof, the polypeptide of SEQ IDNO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a said variant thereof, thepolypeptide of SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a saidvariant thereof, the polypeptide of SEQ ID NO: 78 (Bir16A;AERERIFKRFDANGEGK) or a said variant thereof, and the polypeptide of SEQID NO: 69 (Bir09B; KEMGETLLRAVESYLLAHS) or a said variant thereof; orvi) the polypeptide of SEQ ID NO: 74 (Bir12B; AKYMVIQGEPGRVIRGK) or asaid variant thereof, the polypeptide of SEQ ID NO: 52 (Bir02I;PAARMFKAFILD) or a said variant thereof, the polypeptide of SEQ ID NO:48 (Bir01I; FNYETETTSVIPAARK) or a said variant thereof, the polypeptideof SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV) or a said variant thereof,the polypeptide of SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK) or a saidvariant thereof, and the polypeptide of SEQ ID NO: 78 (Bir16A;AERERIFKRFDANGEGK).